U.S. patent number 8,278,507 [Application Number 12/789,259] was granted by the patent office on 2012-10-02 for polypeptides having cellobiohydrolase activity and polynucleotides encoding same.
This patent grant is currently assigned to Novozymes, A/S, Novozymes, Inc.. Invention is credited to Paul Harris, Ye Liu, Lan Tang, Wenping Wu.
United States Patent |
8,278,507 |
Liu , et al. |
October 2, 2012 |
Polypeptides having cellobiohydrolase activity and polynucleotides
encoding same
Abstract
The present invention relates to isolated polypeptides having
cellobiohydrolase activity and isolated polynucleotides encoding
the polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods of producing and using the polypeptides.
Inventors: |
Liu; Ye (Beijing,
CN), Tang; Lan (Beijing, CN), Harris;
Paul (Carnation, WA), Wu; Wenping (Beijing,
CN) |
Assignee: |
Novozymes, Inc. (Davis, CA)
Novozymes, A/S (Bagsvaerd, DK)
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Family
ID: |
42320866 |
Appl.
No.: |
12/789,259 |
Filed: |
May 27, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100306879 A1 |
Dec 2, 2010 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61183490 |
Jun 2, 2009 |
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Current U.S.
Class: |
800/284; 800/285;
536/23.7; 536/23.2; 435/320.1; 536/23.1; 800/295; 800/278;
435/468 |
Current CPC
Class: |
C12Y
302/01099 (20130101); C12N 15/1137 (20130101); C12N
1/22 (20130101); C12P 7/10 (20130101); C12P
19/02 (20130101); C12N 9/2437 (20130101); C12Y
302/01091 (20130101); C12P 19/14 (20130101); C12N
2310/14 (20130101); Y02E 50/10 (20130101); Y02E
50/16 (20130101) |
Current International
Class: |
C12N
15/82 (20060101); A01H 5/00 (20060101); C12N
15/00 (20060101); C12N 15/09 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2004/056981 |
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Jul 2004 |
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WO |
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2006074435 |
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Jul 2006 |
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WO |
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WO 2008/095033 |
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Aug 2008 |
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WO |
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2009042871 |
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Apr 2009 |
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WO |
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Other References
Liu et al., Uniprot Database, XP-00252514, 2005. cited by
other.
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Primary Examiner: Page; Brent T
Attorney, Agent or Firm: Fechter; Eric J. Starnes; Robert
L.
Government Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
This invention was made with Government support under Cooperative
Agreement DE-FC36-08GO18080 awarded by the Department of Energy.
The government has certain rights in this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application
No. 61/183,490, filed Jun. 2, 2009, which application is
incorporated herein by reference.
Claims
What is claimed is:
1. An isolated polypeptide having cellobiohydrolase activity,
selected from the group consisting of: (a) a polypeptide having at
least 90% sequence identity to the mature polypeptide of SEQ ID NO:
2 or SEQ ID NO: 4; (b) a polypeptide encoded by a polynucleotide
that hybridizes under high stringency conditions with (i) the
mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
(ii) the cDNA sequence contained in the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) the full-length
complementary strand of (i) or (ii); and (c) a polypeptide encoded
by a polynucleotide having at least 90% sequence identity to the
mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:
3.
2. The polypeptide of claim 1, which is encoded by the
polynucleotide contained in plasmid pGEM-T-CBHII45097 which is
contained in E. coli DSM 22598 or plasmid pGEM-T-CBHII45178 which
is contained in E. coli DSM 22599.
3. A composition comprising the polypeptide of claim 1.
4. An isolated polynucleotide encoding the polypeptide of claim
1.
5. A recombinant host cell comprising the polynucleotide of claim 4
operably linked to one or more control sequences that direct the
production of the polypeptide.
6. A method of producing the polypeptide of claim 1, comprising:
(a) cultivating a cell, which in its wild-type form produces the
polypeptide, under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
7. A method of producing a polypeptide having cellobiohydrolase
activity, comprising: (a) cultivating the host cell of claim 5
under conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.
8. A transgenic plant, plant part or plant cell transformed with a
polynucleotide encoding the polypeptide of claim 1.
9. A method of producing a polypeptide having cellobiohydrolase
activity, comprising: (a) cultivating the transgenic plant or plant
cell of claim 8 under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
10. A method of producing a mutant of a parent cell, comprising
inactivating a polynucleotide encoding the polypeptide of claim 1,
which results in the mutant producing less of the polypeptide than
the parent cell.
11. A double-stranded inhibitory RNA (dsRNA) molecule comprising a
subsequence of the polynucleotide of claim 4, wherein optionally
the dsRNA is an siRNA or an miRNA molecule.
12. A method of inhibiting the expression of a polypeptide having
cellobiohydrolase activity in a cell, comprising administering to
the cell or expressing in the cell the double-stranded inhibitory
RNA (dsRNA) molecule of claim 11.
13. An isolated polynucleotide encoding a signal peptide comprising
or consisting of amino acids 1 to 17 of SEQ ID NO: 2 or amino acids
1 to 17 of SEQ ID NO: 4.
14. A method of producing a protein, comprising: (a) cultivating a
recombinant host cell comprising a gene encoding a protein operably
linked to the polynucleotide of claim 13, wherein the gene is
foreign to the polynucleotide encoding the signal peptide, under
conditions conducive for production of the protein; and (b)
recovering the protein.
15. A method for degrading or converting a cellulosic material,
comprising: treating the cellulosic material with an enzyme
composition in the presence of the polypeptide having
cellobiohydrolase activity of claim 1.
16. The method of claim 15, further comprising recovering the
degraded cellulosic material.
17. A method for producing a fermentation product, comprising: (a)
saccharifying a cellulosic material with an enzyme composition in
the presence of the polypeptide having cellobiohydrolase activity
of claim 1; (b) fermenting the saccharified cellulosic material
with one or more fermenting microorganisms to produce the
fermentation product; and (c) recovering the fermentation product
from the fermentation.
18. A method of fermenting a cellulosic material, comprising:
fermenting the cellulosic material with one or more fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition in the presence of a polypeptide having
cellobiohydrolase activity of claim 1.
19. The method of claim 18, wherein the fermenting of the
cellulosic material produces a fermentation product.
20. The method of claim 19, further comprising recovering the
fermentation product from the fermentation.
21. The polypeptide of claim 1, having at least 90% sequence
identity to the mature polypeptide of SEQ ID NO: 2.
22. The polypeptide of claim 1, having at least 95% sequence
identity to the mature polypeptide of SEQ ID NO: 2.
23. The polypeptide of claim 1, having at least 97% sequence
identity to the mature polypeptide of SEQ ID NO: 2.
24. The polypeptide of claim 1, having at least 98% sequence
identity to the mature polypeptide of SEQ ID NO: 2.
25. The polypeptide of claim 1, having at least 99% sequence
identity to the mature polypeptide of SEQ ID NO: 2.
26. The polypeptide of claim 1, comprising or consisting of SEQ ID
NO: 2.
27. The polypeptide of claim 1, comprising or consisting of the
mature polypeptide of SEQ ID NO: 2.
28. The polypeptide of claim 27, wherein the mature polypeptide is
amino acids 18 to 493 of SEQ ID NO: 2.
29. The polypeptide of claim 1, which is encoded by a
polynucleotide that hybridizes under high stringency conditions
with (i) the mature polypeptide coding sequence of SEQ ID NO: 1,
(ii) the cDNA sequence contained in the mature polypeptide coding
sequence of SEQ ID NO: 1, or (iii) the full-length complementary
strand of (i) or (ii).
30. The polypeptide of claim 1, which is encoded by a
polynucleotide that hybridizes under very high stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1, (ii) the cDNA sequence contained in the mature
polypeptide coding sequence of SEQ ID NO: 1, or (iii) the
full-length complementary strand of (i) or (ii).
31. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 90% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1; or the cDNA sequence
thereof.
32. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 95% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1; or the cDNA sequence
thereof.
33. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 97% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1; or the cDNA sequence
thereof.
34. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 98% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1; or the cDNA sequence
thereof.
35. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 99% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1; or the cDNA sequence
thereof.
36. The polypeptide of claim 1, which is encoded by a
polynucleotide comprising or consisting of SEQ ID NO: 1; or the
cDNA sequence thereof.
37. The polypeptide of claim 1, which is encoded by a
polynucleotide comprising or consisting of the mature polypeptide
coding sequence of SEQ ID NO: 1; or the cDNA sequence thereof.
38. The polypeptide of claim 37, wherein the mature polypeptide
coding sequence is nucleotides 52 to 1595 of SEQ ID NO: 1.
39. A nucleic acid construct or expression vector comprising a gene
encoding a protein operably linked to the polynucleotide of claim
13, wherein the gene is foreign to the polynucleotide encoding the
signal peptide.
40. The polypeptide of claim 1, having at least 90% sequence
identity to the mature polypeptide of SEQ ID NO: 4.
41. The polypeptide of claim 1, having at least 95% sequence
identity to the mature polypeptide of SEQ ID NO: 4.
42. The polypeptide of claim 1, having at least 97% sequence
identity to the mature polypeptide of SEQ ID NO: 4.
43. The polypeptide of claim 1, having at least 98% sequence
identity to the mature polypeptide of SEQ ID NO: 4.
44. The polypeptide of claim 1, having at least 99% sequence
identity to the mature polypeptide of SEQ ID NO: 4.
45. The polypeptide of claim 1, comprising or consisting of SEQ ID
NO: 4.
46. The polypeptide of claim 1, comprising or consisting of the
mature polypeptide of SEQ ID NO: 4.
47. The polypeptide of claim 46, wherein the mature polypeptide is
amino acids 18 to 487 of SEQ ID NO: 4.
48. The polypeptide of claim 1, which is encoded by a
polynucleotide that hybridizes under high stringency conditions
with (i) the mature polypeptide coding sequence of SEQ ID NO: 3,
(ii) the cDNA sequence contained in the mature polypeptide coding
sequence of SEQ ID NO: 3, or (iii) the full-length complementary
strand of (i) or (ii).
49. The polypeptide of claim 1, which is encoded by a
polynucleotide that hybridizes under very high stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 3, (ii) the cDNA sequence contained in the mature
polypeptide coding sequence of SEQ ID NO: 3, or (iii) the
full-length complementary strand of (i) or (ii).
50. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 90% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 3; or the cDNA sequence
thereof.
51. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 95% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 3; or the cDNA sequence
thereof.
52. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 97% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 3; or the cDNA sequence
thereof.
53. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 98% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 3; or the cDNA sequence
thereof.
54. The polypeptide of claim 1, which is encoded by a
polynucleotide having at least 99% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 3; or the cDNA sequence
thereof.
55. The polypeptide of claim 1, which is encoded by a
polynucleotide comprising or consisting of SEQ ID NO: 3; or the
cDNA sequence thereof.
56. The polypeptide of claim 1, which is encoded by a
polynucleotide comprising or consisting of the mature polypeptide
coding sequence of SEQ ID NO: 3; or the cDNA sequence thereof.
57. The polypeptide of claim 56, wherein the mature polypeptide
coding sequence is nucleotides 52 to 1581 of SEQ ID NO: 3.
Description
REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable
form, which is incorporated herein by reference.
REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL
This application contains a reference to a deposit of biological
material, which deposit is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to polypeptides having
cellobiohydrolase activity and polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods of producing and using the polypeptides.
2. Description of the Related Art
Cellulose is a polymer of the simple sugar glucose linked by
beta-1,4 bonds. Many microorganisms produce enzymes that hydrolyze
beta-linked glucans. These enzymes include endoglucanases,
cellobiohydrolases, and beta-glucosidases. Endoglucanases digest
the cellulose polymer at random locations, opening it to attack by
cellobiohydrolases. Cellobiohydrolases sequentially release
molecules of cellobiose from the ends of the cellulose polymer.
Cellobiose is a water-soluble beta-1,4-linked dimer of glucose.
Beta-glucosidases hydrolyze cellobiose to glucose.
The conversion of lignocellulosic feedstocks into ethanol has the
advantages of the ready availability of large amounts of feedstock,
the desirability of avoiding burning or land filling the materials,
and the cleanliness of the ethanol fuel. Wood, agricultural
residues, herbaceous crops, and municipal solid wastes have been
considered as feedstocks for ethanol production. These materials
primarily consist of cellulose, hemicellulose, and lignin. Once the
cellulose is converted to glucose, the glucose is easily fermented
by yeast into ethanol.
There is a need in the art to improve cellulolytic protein
compositions through supplementation with additional enzymes to
increase efficiency and to provide cost-effective enzyme solutions
for degradation of lignocellulose.
The present invention provides polypeptides having
cellobiohydrolase activity and polynucleotides encoding the
polypeptides.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having
cellobiohydrolase activity selected from the group consisting
of:
(a) a polypeptide having at least 90% sequence identity to the
mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;
(b) a polypeptide encoded by a polynucleotide that hybridizes under
high stringency conditions or very high stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ
ID NO: 3, (ii) the cDNA sequence contained in the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or
(iii) the full-length complementary strand of (i) or (ii);
(c) a polypeptide encoded by a polynucleotide having at least 90%
sequence identity to the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3;
(d) a variant comprising a substitution, deletion, and/or insertion
of one or more (several) amino acids of the mature polypeptide of
SEQ ID NO: 2 or SEQ ID NO: 4; and
(e) a fragment of the polypeptide of (a), (b), (c), or (d) that has
cellobiohydrolase activity.
The present invention also relates to isolated polynucleotides
encoding the polypeptides of the present invention; nucleic acid
constructs, recombinant expression vectors, and recombinant host
cells comprising the polynucleotides; and methods of producing the
polypeptides.
The present invention also relates to methods for degrading or
converting a cellulosic material, comprising: treating the
cellulosic material with an enzyme composition in the presence of a
polypeptide having cellobiohydrolase activity of the present
invention.
The present invention also relates to methods for producing a
fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition
in the presence of a polypeptide having cellobiohydrolase activity
of the present invention;
(b) fermenting the saccharified cellulosic material with one or
more fermenting microorganisms to produce the fermentation product;
and
(c) recovering the fermentation product from the fermentation.
The present invention also relates to methods of fermenting a
cellulosic material, comprising: fermenting the cellulosic material
with one or more fermenting microorganisms, wherein the cellulosic
material is saccharified with an enzyme composition in the presence
of a polypeptide having cellobiohydrolase activity of the present
invention.
The present invention also relates to a polynucleotide encoding a
signal peptide comprising or consisting of amino acids 1 to 17 of
SEQ ID NO: 2 or amino acids 1 to 17 of SEQ ID NO: 4, which is
operably linked to a gene encoding a protein; nucleic acid
constructs, expression vectors, and recombinant host cells
comprising the polynucleotides; and methods of producing a
protein.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the genomic DNA sequence and the deduced amino acid
sequence of a Thielavia hyrcaniae NN045097 Family 6
cellobiohydrolase gene (SEQ ID NOs: 1 and 2, respectively).
FIG. 2 shows the genomic DNA sequence and the deduced amino acid
sequence of a Thielavia hyrcaniae NN045178 Family 6
cellobiohydrolase gene (SEQ ID NOs: 3 and 4, respectively).
FIG. 3 shows a restriction map of pCBHII45178.
FIG. 4 shows a restriction map of pGEM-T-CBHII45178.
FIG. 5 shows a restriction map of pCBHOII45097.
FIG. 6 shows a restriction map of pGEM-T-CBHII45097.
FIG. 7 shows the pH activity profiles of the Thielavia hyrcaniae
NN045097 and NN045178 cellobiohydrolases at 50.degree. C.
FIGS. 8A and 8B show the stability of the Thielavia hyrcaniae
NN045097 and NN045178 cellobiohydrolases as a function of pH at
50.degree. C.
FIG. 9 shows the temperature activity profiles of the Thielavia
hyrcaniae NN045097 and NN045178 cellobiohydrolases at pH 5.
FIGS. 10A and 10B show the stability of the Thielavia hyrcaniae
NN045097 and NN045178 cellobiohydrolases as a function of
temperature at pH 5.
DEFINITIONS
Cellobiohydrolase: The term "cellobiohydrolase" means a
1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which
catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in
cellulose, cellooligosaccharides, or any beta-1,4-linked glucose
containing polymer, releasing cellobiose from the reducing or
non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose
degradation: New insight into the function of cellobiohydrolases,
Trends in Biotechnology 15: 160-167; Teeri et al., 1998,
Trichoderma reesei cellobiohydrolases: why so efficient on
crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178). For
purposes of the present invention, cellobiohydrolase activity is
determined according to the procedures described by Lever et al.,
1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS
Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS
Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem.
170: 575-581. The Lever et al. method can be employed to assess
hydrolysis of cellulose in corn stover, while the methods of van
Tilbeurgh et al. and Tomme et al. can be used to determine the
cellobiohydrolase activity on a fluorescent disaccharide
derivative, 4-methylumbelliferyl-.beta.-D-lactoside. In addition,
PASC can be used as a substrate as described herein.
The polypeptides of the present invention have at least 20%, e.g.,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 95%, and at least 100% of the
cellobiohydrolase activity of the mature polypeptide of SEQ ID NO:
2 or SEQ ID NO: 4.
Family 6, GH6, or CEL6: The terms "Family 6", "GH6", or "CEL6" are
defined herein as a polypeptide falling into the glycoside
hydrolase Family 6, according to Henrissat B., 1991, A
classification of glycosyl hydrolases based on amino-acid sequence
similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch,
1996, Updating the sequence-based classification of glycosyl
hydrolases, Biochem. J. 316: 695-696.
Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or
"cellulase" means one or more (several) enzymes that hydrolyze a
cellulosic material. Such enzymes include endoglucanase(s),
cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
The two basic approaches for measuring cellulolytic activity
include: (1) measuring the total cellulolytic activity, and (2)
measuring the individual cellulolytic activities (endoglucanases,
cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et
al., Outlook for cellulase improvement: Screening and selection
strategies, 2006, Biotechnology Advances 24: 452-481. Total
cellulolytic activity is usually measured using insoluble
substrates, including Whatman No 1 filter paper, microcrystalline
cellulose, bacterial cellulose, algal cellulose, cotton, pretreated
lignocellulose, etc. The most common total cellulolytic activity
assay is the filter paper assay using Whatman No 1 filter paper as
the substrate. The assay was established by the International Union
of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of
cellulase activities, Pure Appl. Chem. 59: 257-68).
For purposes of the present invention, cellulolytic enzyme activity
is determined by measuring the increase in hydrolysis of a
cellulosic material by cellulolytic enzyme(s) under the following
conditions: 1-20 mg of cellulolytic enzyme protein/g of cellulose
in PCS for 3-7 days at 50.degree. C. compared to a control
hydrolysis without addition of cellulolytic enzyme protein. Typical
conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble
solids, 50 mM sodium acetate pH 5, 1 mM MnSO.sub.4, 50-65.degree.
C., 72 hours, sugar analysis by AMINEX.RTM. HPX-87H column (Bio-Rad
Laboratories, Inc., Hercules, Calif., USA).
Endoglucanase: The term "endoglucanase" means an
endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),
which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages in
cellulose, cellulose derivatives (such as carboxymethyl cellulose
and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed
beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and
other plant material containing cellulosic components.
Endoglucanase activity can be determined by measuring reduction in
substrate viscosity or increase in reducing ends determined by a
reducing sugar assay (Zhang et al., 2006, Biotechnology Advances
24: 452-481). For purposes of the present invention, endoglucanase
activity is determined using carboxymethyl cellulose (CMC) as
substrate according to the procedure of Ghose, 1987, Pure and Appl.
Chem. 59: 257-268, at pH 5, 40.degree. C.
Beta-glucosidase: The term "beta-glucosidase" means a
beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) activity that
catalyzes the hydrolysis of terminal non-reducing beta-D-glucose
residues with the release of beta-D-glucose. Cellobiase is
synonymous with beta-glucosidase. For purposes of the present
invention, beta-glucosidase activity is determined at 25.degree. C.
using 1 mM 4-nitrophenyl-beta-D-glucopyranoside as substrate in 50
mM sodium citrate pH 4.8. One unit of beta-glucosidase activity is
defined as 1.0 pmole of p-nitrophenolate anion produced per minute
at 25.degree. C., pH 4.8.
Cellulolytic enhancing activity: The term "cellulolytic enhancing
activity" means a biological activity catalyzed by a GH61
polypeptide that enhances the hydrolysis of a cellulosic material
by enzyme having cellulolytic activity. For purposes of the present
invention, cellulolytic enhancing activity is determined by
measuring the increase in reducing sugars or the increase of the
total of cellobiose and glucose from the hydrolysis of a cellulosic
material by cellulolytic enzyme under the following conditions:
1-50 mg of total protein/g of cellulose in PCS, wherein total
protein is comprised of 50-99.5% w/w cellulolytic enzyme protein
and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic
enhancing activity for 1-7 days at 50.degree. C. compared to a
control hydrolysis with equal total protein loading without
cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g
of cellulose in PCS). In a preferred aspect, a mixture of
CELLUCLAST.RTM. 1.5L (Novozymes A/S, Bagsvrd, Denmark) in the
presence of 2-3% of total protein weight Aspergillus oryzae
beta-glucosidase (recombinantly produced in Aspergillus oryzae
according to WO 02/095014) or 3% of total protein weight
Aspergillus fumigatus beta-glucosidase (recombinantly produced in
Aspergillus oryzae as described in WO 2002/095014) of cellulase
protein loading is used as the source of the cellulolytic
activity.
The GH61 polypeptides having cellulolytic enhancing activity
enhance the hydrolysis of a cellulosic material catalyzed by enzyme
having cellulolytic activity by reducing the amount of cellulolytic
enzyme required to reach the same degree of hydrolysis preferably
at least 1.01-fold, more preferably at least 1.05-fold, more
preferably at least 1.10-fold, more preferably at least 1.25-fold,
more preferably at least 1.5-fold, more preferably at least 2-fold,
more preferably at least 3-fold, more preferably at least 4-fold,
more preferably at least 5-fold, even more preferably at least
10-fold, and most preferably at least 20-fold.
Family 61 glycoside hydrolase: The term "Family 61 glycoside
hydrolase" or "Family GH61" or "GH61" means a polypeptide falling
into the glycoside hydrolase Family 61 according to Henrissat B.,
1991, A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B.,
and Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696.
Hemicellulolytic enzyme or hemicellulase: The term
"hemicellulolytic enzyme" or "hemicellulase" means one or more
(several) enzymes that hydrolyze a hemicellulosic material. See,
for example, Shallom, D. and Shoham, Y. Microbial hemicellulases.
Current Opinion In Microbiology, 2003, 6(3): 219-228).
Hemicellulases are key components in the degradation of plant
biomass. Examples of hemicellulases include, but are not limited
to, an acetylmannan esterase, an acetyxylan esterase, an
arabinanase, an arabinofuranosidase, a coumaric acid esterase, a
feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl
esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
The substrates of these enzymes, the hemicelluloses, are a
heterogeneous group of branched and linear polysaccharides that are
bound via hydrogen bonds to the cellulose microfibrils in the plant
cell wall, crosslinking them into a robust network. Hemicelluloses
are also covalently attached to lignin, forming together with
cellulose a highly complex structure. The variable structure and
organization of hemicelluloses require the concerted action of many
enzymes for its complete degradation. The catalytic modules of
hemicellulases are either glycoside hydrolases (GHs) that hydrolyze
glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze
ester linkages of acetate or ferulic acid side groups. These
catalytic modules, based on homology of their primary sequence, can
be assigned into GH and CE families marked by numbers. Some
families, with overall similar fold, can be further grouped into
clans, marked alphabetically (e.g., GH-A). A most informative and
updated classification of these and other carbohydrate active
enzymes is available on the Carbohydrate-Active Enzymes (CAZy)
database. Hemicellulolytic enzyme activities can be measured
according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:
1739-1752.
Xylan degrading activity or xylanolytic activity: The term "xylan
degrading activity" or "xylanolytic activity" means a biological
activity that hydrolyzes xylan-containing material. The two basic
approaches for measuring xylanolytic activity include: (1)
measuring the total xylanolytic activity, and (2) measuring the
individual xylanolytic activities (e.g., endoxylanases,
beta-xylosidases, arabinofuranosidases, alpha-glucuronidases,
acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl
esterases). Recent progress in assays of xylanolytic enzymes was
summarized in several publications including Biely and Puchard,
Recent progress in the assays of xylanolytic enzymes, 2006, Journal
of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova
and Biely, 2006, Glucuronoyl esterase--Novel carbohydrate esterase
produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601;
Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997,
The beta-D-xylosidase of Trichoderma reesei is a multifunctional
beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
Total xylan degrading activity can be measured by determining the
reducing sugars formed from various types of xylan, including, for
example, oat spelt, beechwood, and larchwood xylans, or by
photometric determination of dyed xylan fragments released from
various covalently dyed xylans. The most common total xylanolytic
activity assay is based on production of reducing sugars from
polymeric 4-O-methyl glucuronoxylan as described in Bailey, Biely,
Poutanen, 1992, Interlaboratory testing of methods for assay of
xylanase activity, Journal of Biotechnology 23(3): 257-270.
For purposes of the present invention, xylan degrading activity is
determined by measuring the increase in hydrolysis of birchwood
xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by
xylan-degrading enzyme(s) under the following typical conditions: 1
ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic
protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C.,
24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide
(PHBAH) assay as described by Lever, 1972, A new reaction for
colorimetric determination of carbohydrates, Anal. Biochem 47:
273-279.
Xylanase: The term "xylanase" means a
1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the
endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined
with 0.2% AZCL-arabinoxylan as substrate in 0.01% Triton X-100 and
200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit of
xylanase activity is defined as 1.0 .mu.mole of azurine produced
per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as
substrate in 200 mM sodium phosphate pH 6 buffer.
Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside
xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of
short beta (1.fwdarw.4)-xylooligosaccharides, to remove successive
D-xylose residues from the non-reducing termini. For purposes of
the present invention, one unit of beta-xylosidase is defined as
1.0 .mu.mole of p-nitrophenolate anion produced per minute at
40.degree. C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as
substrate in 100 mM sodium citrate containing 0.01% TWEEN.RTM.
20.
Acetylxylan esterase: The term "acetylxylan esterase" means a
carboxylesterase (EC 3.1.1.72) that catalyses the hydrolysis of
acetyl groups from polymeric xylan, acetylated xylose, acetylated
glucose, alpha-napthyl acetate, and p-nitrophenylacetate. For
purposes of the present invention, acetylxylan esterase activity is
determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM
sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20. One unit of
acetylxylan esterase is defined as the amount of enzyme capable of
releasing 1 .mu.mole of p-nitrophenolate anion per minute at pH 5,
25.degree. C.
Feruloyl esterase: The term "feruloyl esterase" means a
4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that
catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl
(feruloyl) group from an esterified sugar, which is usually
arabinose in "natural" substrates, to produce ferulate
(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as
ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III,
cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For
purposes of the present invention, feruloyl esterase activity is
determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM
sodium acetate pH 5.0. One unit of feruloyl esterase equals the
amount of enzyme capable of releasing 1 pmole of p-nitrophenolate
anion per minute at pH 5, 25.degree. C.
Alpha-glucuronidase: The term "alpha-glucuronidase" means an
alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that
catalyzes the hydrolysis of an alpha-D-glucuronoside to
D-glucuronate and an alcohol. For purposes of the present
invention, alpha-glucuronidase activity is determined according to
de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of
alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 .mu.mole of glucuronic or 4-0-methylglucuronic acid per
minute at pH 5, 40.degree. C.
Alpha-L-arabinofuranosidase: The term "alpha-L-arabinofuranosidase"
means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC
3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing
alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The
enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans
containing (1,3)- and/or (1,5)-linkages, arabinoxylans, and
arabinogalactans. Alpha-L-arabinofuranosidase is also known as
arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,
alpha-arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase, or alpha-L-arabinanase. For purposes of the
present invention, alpha-L-arabinofuranosidase activity is
determined using 5 mg of medium viscosity wheat arabinoxylan
(Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland)
per ml of 100 mM sodium acetate pH 5 in a total volume of 200 .mu.l
for 30 minutes at 40.degree. C. followed by arabinose analysis by
AMINEX.RTM. HPX-87H column chromatography (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA).
Cellulosic material: The cellulosic material can be any material
containing cellulose.
The predominant polysaccharide in the primary cell wall of biomass
is cellulose, the second most abundant is hemicellulose, and the
third is pectin. The secondary cell wall, produced after the cell
has stopped growing, also contains polysaccharides and is
strengthened by polymeric lignin covalently cross-linked to
hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and
thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a
variety of compounds, such as xylans, xyloglucans, arabinoxylans,
and mannans in complex branched structures with a spectrum of
substituents. Although generally polymorphous, cellulose is found
in plant tissue primarily as an insoluble crystalline matrix of
parallel glucan chains. Hemicelluloses usually hydrogen bond to
cellulose, as well as to other hemicelluloses, which help stabilize
the cell wall matrix.
Cellulose is generally found, for example, in the stems, leaves,
hulls, husks, and cobs of plants or leaves, branches, and wood of
trees. The cellulosic material can be, but is not limited to,
herbaceous material, agricultural residue, forestry residue,
municipal solid waste, waste paper, and pulp and paper mill residue
(see, for example, Wiselogel et al., 1995, in Handbook on
Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor &
Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50:
3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25:
695-719; Mosier et al., 1999, Recent Progress in Bioconversion of
Lignocellulosics, in Advances in Biochemical
Engineering/Biotechnology, T. Scheper, managing editor, Volume 65,
pp.23-40, Springer-Verlag, New York). It is understood herein that
the cellulose may be in the form of lignocellulose, a plant cell
wall material containing lignin, cellulose, and hemicellulose in a
mixed matrix. In a preferred aspect, the cellulosic material is
lignocelluloses, which comprises cellulose, hemicellulose, and
lignin.
In one aspect, the cellulosic material is herbaceous material. In
another aspect, the cellulosic material is agricultural residue. In
another aspect, the cellulosic material is forestry residue. In
another aspect, the cellulosic material is municipal solid waste.
In another aspect, the cellulosic material is waste paper. In
another aspect, the cellulosic material is pulp and paper mill
residue.
In another aspect, the cellulosic material is corn stover. In
another aspect, the cellulosic material is corn fiber. In another
aspect, the cellulosic material is corn cob. In another aspect, the
cellulosic material is orange peel. In another aspect, the
cellulosic material is rice straw. In another aspect, the
cellulosic material is wheat straw. In another aspect, the
cellulosic material is switch grass. In another aspect, the
cellulosic material is miscanthus. In another aspect, the
cellulosic material is bagasse.
In another aspect, the cellulosic material is microcrystalline
cellulose. In another aspect, the cellulosic material is bacterial
cellulose. In another aspect, the cellulosic material is algal
cellulose. In another aspect, the cellulosic material is cotton
linter. In another aspect, the cellulosic material is amorphous
phosphoric-acid treated cellulose. In another aspect, the
cellulosic material is filter paper.
The cellulosic material may be used as is or may be subjected to
pretreatment, using conventional methods known in the art, as
described herein. In a preferred aspect, the cellulosic material is
pretreated.
Pretreated corn stover: The term "PCS" or "Pretreated Corn Stover"
means a cellulosic material derived from corn stover by treatment
with heat and dilute sulfuric acid. Isolated or Purified: The term
"isolated" or "purified" means a polypeptide or polynucleotide that
is removed from at least one component with which it is naturally
associated. For example, a polypeptide may be at least 1% pure,
e.g., at least 5% pure, at least 10% pure, at least 20% pure, at
least 40% pure, at least 60% pure, at least 80% pure, at least 90%
pure, or at least 95% pure, as determined by SDS-PAGE and a
polynucleotide may be at least 1% pure, e.g., at least 5% pure, at
least 10% pure, at least 20% pure, at least 40% pure, at least 60%
pure, at least 80% pure, at least 90% pure, or at least 95% pure,
as determined by agarose electrophoresis.
Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. In one
aspect, the mature polypeptide is amino acids 18 to 493 of SEQ ID
NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein
Engineering 10: 1-6) that predicts amino acids 1 to 17 of SEQ ID
NO: 2 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 18 to 487 of SEQ ID NO: 4 based on the
SignalP program (Nielsen et al., 1997, supra) that predicts amino
acids 1 to 17 of SEQ ID NO: 4 are a signal peptide. It is known in
the art that a host cell may produce a mixture of two of more
different mature polypeptides (i.e., with a different C-terminal
and/or N-terminal amino acid) expressed by the same
polynucleotide.
Mature polypeptide coding sequence: The term "mature polypeptide
coding sequence" means a polynucleotide that encodes a mature
polypeptide having cellobiohydrolase activity. In one aspect, the
mature polypeptide coding sequence is nucleotides 52 to 1595 of SEQ
ID NO: 1 based on the SignalP program (Nielsen et al., 1997, supra)
that predicts nucleotides 1 to 51 of SEQ ID NO: 1 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 52 to 1581 of SEQ ID NO: 3 based on the SignalP
program (Nielsen et al., 1997, supra) that predicts nucleotides 1
to 51 of SEQ ID NO: 3 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is the cDNA sequence
contained in nucleotides 52 to 1595 of SEQ ID NO: 1. In another
aspect, the mature polypeptide coding sequence is the cDNA sequence
contained in nucleotides 52 to 1581 of SEQ ID NO: 3.
Sequence Identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"sequence identity".
For purposes of the present invention, the degree of sequence
identity between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 3.0.0 or later. The optional parameters used are
gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The
output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows: (Identical Residues.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
For purposes of the present invention, the degree of sequence
identity between two deoxyribonucleotide sequences is determined
using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,
supra) as implemented in the Needle program of the EMBOSS package
(EMBOSS: The European Molecular Biology Open Software Suite, Rice
et al., 2000, supra), preferably version 3.0.0 or later. The
optional parameters used are gap open penalty of 10, gap extension
penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4)
substitution matrix. The output of Needle labeled "longest
identity" (obtained using the -nobrief option) is used as the
percent identity and is calculated as follows: (Identical
Deoxyribonucleotides.times.100)/(Length of Alignment-Total Number
of Gaps in Alignment)
Fragment: The term "fragment" means a polypeptide having one or
more (several) amino acids deleted from the amino and/or carboxyl
terminus of a mature polypeptide; wherein the fragment has
cellobiohydrolase activity. In one aspect, a fragment contains at
least 400 amino acid residues, more preferably at least 425 amino
acid residues, and most preferably at least 450 amino acid
residues, of the mature polypeptide of SEQ ID NO: 2. In another
aspect, a fragment contains at least 400 amino acid residues, more
preferably at least 425 amino acid residues, and most preferably at
least 450 amino acid residues, of the mature polypeptide of SEQ ID
NO: 4.
Subsequence: The term "subsequence" means a polynucleotide having
one or more (several) nucleotides deleted from the 5' and/or 3' end
of a mature polypeptide coding sequence; wherein the subsequence
encodes a fragment having cellobiohydrolase activity. In one
aspect, a subsequence contains at least 1200 nucleotides, more
preferably at least 1275 nucleotides, and most preferably at least
1350 nucleotides of the mature polypeptide coding sequence of SEQ
ID NO: 1. In another aspect, a subsequence contains at least 1200
nucleotides, more preferably at least 1275 nucleotides, and most
preferably at least 1350 nucleotides of the mature polypeptide
coding sequence of SEQ ID NO: 3.
Allelic variant: The term "allelic variant" means any of two or
more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
Coding sequence: The term "coding sequence" means a polynucleotide,
which directly specifies the amino acid sequence of a polypeptide.
The boundaries of the coding sequence are generally determined by
an open reading frame, which usually begins with the ATG start
codon or alternative start codons such as GTG and TTG and ends with
a stop codon such as TAA, TAG, and TGA. The coding sequence may be
a DNA, cDNA, synthetic, or recombinant polynucleotide.
cDNA: The term "cDNA" means a DNA molecule that can be prepared by
reverse transcription from a mature, spliced, mRNA molecule
obtained from a eukaryotic cell. cDNA lacks intron sequences that
may be present in the corresponding genomic DNA. The initial,
primary RNA transcript is a precursor to mRNA that is processed
through a series of steps, including splicing, before appearing as
mature spliced mRNA.
Nucleic acid construct: The term "nucleic acid construct" means a
nucleic acid molecule, either single- or double-stranded, which is
isolated from a naturally occurring gene or is modified to contain
segments of nucleic acids in a manner that would not otherwise
exist in nature or which is synthetic. The term nucleic acid
construct is synonymous with the term "expression cassette" when
the nucleic acid construct contains the control sequences required
for expression of a coding sequence of the present invention.
Control sequences: The term "control sequences" means all
components necessary for the expression of a polynucleotide
encoding a polypeptide of the present invention. Each control
sequence may be native or foreign to the polynucleotide encoding
the polypeptide or native or foreign to each other. Such control
sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal
peptide sequence, and transcription terminator. At a minimum, the
control sequences include a promoter, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences with the
coding region of the polynucleotide encoding a polypeptide.
Operably linked: The term "operably linked" means a configuration
in which a control sequence is placed at an appropriate position
relative to the coding sequence of a polynucleotide such that the
control sequence directs the expression of the coding sequence.
Expression: The term "expression" includes any step involved in the
production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
Expression vector: The term "expression vector" means a linear or
circular DNA molecule that comprises a polynucleotide encoding a
polypeptide and is operably linked to additional nucleotides that
provide for its expression.
Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, and the
like with a nucleic acid construct or expression vector comprising
a polynucleotide of the present invention. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication.
Variant: The term "variant" means a polypeptide having
cellobiohydrolase activity comprising an alteration, i.e., a
substitution, insertion, and/or deletion of one or more (several)
amino acid residues at one or more (several) positions. A
substitution means a replacement of an amino acid occupying a
position with a different amino acid; a deletion means removal of
an amino acid occupying a position; and an insertion means adding
one or more (several) amino acids, e.g., 1-5 amino acids, adjacent
to an amino acid occupying a position.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Cellobiohydrolase Activity
The present invention relates to isolated polypeptides having
cellobiohydrolase activity selected from the group consisting
of:
(a) a polypeptide having at least 90% sequence identity to the
mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;
(b) a polypeptide encoded by a polynucleotide that hybridizes under
high stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence
contained in the mature polypeptide coding sequence of SEQ ID NO: 1
or SEQ ID NO: 3, or (iii) the full-length complementary strand of
(i) or (ii);
(c) a polypeptide encoded by a polynucleotide having at least 90%
sequence identity to the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3 or the cDNA sequence thereof;
(d) a variant comprising a substitution, deletion, and/or insertion
of one or more (several) amino acids of the mature polypeptide of
SEQ ID NO: 2 or SEQ ID NO: 4; and
(e) a fragment of a polypeptide of (a), (b), (c), or (d) that has
cellobiohydrolase activity.
The present invention relates to isolated polypeptides having a
sequence identity to the mature polypeptide of SEQ ID NO: 2 or SEQ
ID NO: 4 of at least 90%, e.g., at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100%, which have cellobiohydrolase
activity. In one aspect, the polypeptides differ by no more than
ten amino acids, e.g., by five amino acids, by four amino acids, by
three amino acids, by two amino acids, and by one amino acid from
the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
A polypeptide of the present invention preferably comprises or
consists of the amino acid sequence of SEQ ID NO: 2 or an allelic
variant thereof; or is a fragment thereof having cellobiohydrolase
activity. In another aspect, the polypeptide comprises or consists
of SEQ ID NO: 2. In another aspect, the polypeptide comprises or
consists of the mature polypeptide of SEQ ID NO: 2. In another
preferred aspect, the polypeptide comprises or consists of amino
acids 18 to 493 of SEQ ID NO: 2.
A polypeptide of the present invention also preferably comprises or
consists of the amino acid sequence of SEQ ID NO: 4 or an allelic
variant thereof; or is a fragment thereof having cellobiohydrolase
activity. In another aspect, the polypeptide comprises or consists
of SEQ ID NO: 4. In another aspect, the polypeptide comprises or
consists of the mature polypeptide of
SEQ ID NO: 4. In another preferred aspect, the polypeptide
comprises or consists of amino acids 18 to 487 of SEQ ID NO: 4.
The present invention also relates to isolated polypeptides having
cellobiohydrolase activity that are encoded by polynucleotides that
hybridize under high stringency conditions or very high stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the
mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
or (iii) the full-length complementary strand of (i) or (ii) (J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning,
A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).
The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3 or a subsequence
thereof, as well as the amino acid sequence of SEQ ID NO: 2 or SEQ
ID NO: 4 or a fragment thereof, may be used to design nucleic acid
probes to identify and clone DNA encoding polypeptides having
cellobiohydrolase activity from strains of different genera or
species according to methods well known in the art. In particular,
such probes can be used for hybridization with the genomic or cDNA
of the genus or species of interest, following standard Southern
blotting procedures, in order to identify and isolate the
corresponding gene therein. Such probes can be considerably shorter
than the entire sequence, but should be at least 14, e.g., at least
25, at least 35, or at least 70 nucleotides in length. Preferably,
the nucleic acid probe is at least 100 nucleotides in length, e.g.,
at least 200 nucleotides, at least 300 nucleotides, at least 400
nucleotides, at least 500 nucleotides, at least 600 nucleotides, at
least 700 nucleotides, at least 800 nucleotides, or at least 900
nucleotides in length. Both DNA and RNA probes can be used. The
probes are typically labeled for detecting the corresponding gene
(for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin).
Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may
be screened for DNA that hybridizes with the probes described above
and encodes a polypeptide having cellobiohydrolase activity.
Genomic or other DNA from such other strains may be separated by
agarose or polyacrylamide gel electrophoresis, or other separation
techniques. DNA from the libraries or the separated DNA may be
transferred to and immobilized on nitrocellulose or other suitable
carrier material. In order to identify a clone or DNA that is
homologous with SEQ ID NO: 1 or SEQ ID NO: 3 or a subsequence
thereof, the carrier material is preferably used in a Southern
blot.
For purposes of the present invention, hybridization indicates that
the polynucleotide hybridizes to a labeled nucleic acid probe
corresponding to the mature polypeptide coding sequence of SEQ ID
NO: 1 or SEQ ID NO: 3; the cDNA sequence contained in the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; its
full-length complementary strand; or a subsequence thereof; under
very low to very high stringency conditions. Molecules to which the
nucleic acid probe hybridizes under these conditions can be
detected using, for example, X-ray film.
In one aspect, the nucleic acid probe is the mature polypeptide
coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or the cDNA
sequence thereof. In another aspect, the nucleic acid probe is a
polynucleotide that encodes the polypeptide of SEQ ID NO: 2 or SEQ
ID NO: 4 or a fragment thereof. In another preferred aspect, the
nucleic acid probe is SEQ ID NO: 1 or SEQ ID NO: 3 or the cDNA
sequence thereof. In another aspect, the nucleic acid probe is the
polynucleotide contained in plasmid pGEM-T-CBHII45097 which is
contained in E. coli DSM 22598, wherein the polynucleotide encodes
a polypeptide having cellobiohydrolase activity. In another aspect,
the nucleic acid probe is the mature polypeptide coding region
contained in plasmid pGEM-T-CBHII45097 which is contained in E.
coli DSM 22598. In another aspect, the nucleic acid probe is the
polynucleotide contained in plasmid pGEM-T-CBHII45178 which is
contained in E. coli DSM 22599, wherein the polynucleotide encodes
a polypeptide having cellobiohydrolase activity. In another aspect,
the nucleic acid probe is the mature polypeptide coding region
contained in plasmid pGEM-T-CBHII45178 which is contained in E.
coli DSM 22599.
For long probes of at least 100 nucleotides in length, very low to
very high stringency conditions are defined as prehybridization and
hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and either
25% formamide for very low and low stringencies, 35% formamide for
medium and medium-high stringencies, or 50% formamide for high and
very high stringencies, following standard Southern blotting
procedures for 12 to 24 hours optimally. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 45.degree. C. (very low stringency), at 50.degree. C.
(low stringency), at 55.degree. C. (medium stringency), at
60.degree. C. (medium-high stringency), at 65.degree. C. (high
stringency), and at 70.degree. C. (very high stringency).
For short probes of about 15 nucleotides to about 70 nucleotides in
length, stringency conditions are defined as prehybridization and
hybridization at about 5.degree. C. to about 10.degree. C. below
the calculated T.sub.m using the calculation according to Bolton
and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48:1390) in 0.9 M
NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times.
Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium
monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml
following standard Southern blotting procedures for 12 to 24 hours
optimally. The carrier material is finally washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
The present invention also relates to isolated polypeptides having
cellobiohydrolase activity encoded by polynucleotides having a
sequence identity to the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3 or the cDNA sequence thereof of at least
90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%.
The present invention also relates to variants comprising a
substitution, deletion, and/or insertion of one or more (or
several) amino acids of the mature polypeptide of SEQ ID NO: 2 or
SEQ ID NO: 4, or a homologous sequence thereof. Preferably, amino
acid changes are of a minor nature, that is conservative amino acid
substitutions or insertions that do not significantly affect the
folding and/or activity of the protein; small deletions, typically
of one to about 30 amino acids; small amino- or carboxyl-terminal
extensions, such as an amino-terminal methionine residue; a small
linker peptide of up to about 20-25 residues; or a small extension
that facilitates purification by changing net charge or another
function, such as a poly-histidine tract, an antigenic epitope or a
binding domain.
Examples of conservative substitutions are within the group of
basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. The most commonly occurring
exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
Alternatively, the amino acid changes are of such a nature that the
physico-chemical properties of the polypeptides are altered. For
example, amino acid changes may improve the thermal stability of
the polypeptide, alter the substrate specificity, change the pH
optimum, and the like.
Essential amino acids in a parent polypeptide can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,
1989, Science 244: 1081-1085). In the latter technique, single
alanine mutations are introduced at every residue in the molecule,
and the resultant mutant molecules are tested for cellobiohydrolase
activity to identify amino acid residues that are critical to the
activity of the molecule. See also, Hilton et al., 1996, J. Biol.
Chem. 271: 4699-4708. The active site of the enzyme or other
biological interaction can also be determined by physical analysis
of structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction, or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., 1992, Science 255:
306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver
et al., 1992, FEBS Lett. 309: 59-64. The identities of essential
amino acids can also be inferred from analysis of identities with
polypeptides that are related to the parent polypeptide.
Single or multiple amino acid substitutions, deletions, and/or
insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput,
automated screening methods to detect activity of cloned,
mutagenized polypeptides expressed by host cells (Ness et al.,
1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules
that encode active polypeptides can be recovered from the host
cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
The total number of amino acid substitutions, deletions and/or
insertions of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:
4 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9.
The polypeptide may be hybrid polypeptide in which a portion of one
polypeptide is fused at the N-terminus or the C-terminus of a
portion of another polypeptide.
The polypeptide may be a fused polypeptide or cleavable fusion
polypeptide in which another polypeptide is fused at the N-terminus
or the C-terminus of the polypeptide of the present invention. A
fused polypeptide is produced by fusing a polynucleotide encoding
another polypeptide to a polynucleotide of the present invention.
Techniques for producing fusion polypeptides are known in the art,
and include ligating the coding sequences encoding the polypeptides
so that they are in frame and that expression of the fused
polypeptide is under control of the same promoter(s) and
terminator. Fusion proteins may also be constructed using intein
technology in which fusions are created post-translationally
(Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994,
Science 266: 776-779).
A fusion polypeptide can further comprise a cleavage site between
the two polypeptides. Upon secretion of the fusion protein, the
site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
Sources of Polypeptides Having Cellobiohydrolase Activity
A polypeptide having cellobiohydrolase activity of the present
invention may be obtained from microorganisms of any genus. For
purposes of the present invention, the term "obtained from" as used
herein in connection with a given source shall mean that the
polypeptide encoded by a polynucleotide is produced by the source
or by a strain in which the polynucleotide from the source has been
inserted. In one aspect, the polypeptide obtained from a given
source is secreted extracellularly.
The polypeptide may be a bacterial polypeptide. For example, the
polypeptide may be a gram-positive bacterial polypeptide such as a
Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,
Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or
Streptomyces polypeptide having cellobiohydrolase activity, or a
gram-negative bacterial polypeptide such as a Campylobacter, E.
coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,
Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.
In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis,
or Bacillus thuringiensis polypeptide.
In another aspect, the polypeptide is a Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi
subsp. Zooepidemicus polypeptide.
In another aspect, the polypeptide is a Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, or Streptomyces lividans polypeptide.
The polypeptide may also be a fungal polypeptide. For example, the
polypeptide may be a yeast polypeptide such as a Candida,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia polypeptide; or a filamentous fungal polypeptide such as
an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,
Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium,
Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus,
Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,
Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,
Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus,
Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.
In another aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis
polypeptide.
In another aspect, the polypeptide is an Acremonium cellulolyticus,
Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,
Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium
queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum,
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,
Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium
sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium
venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,
Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora
crassa, Penicillium funiculosum, Penicillium purpurogenum,
Phanerochaete chrysosporium, Thielavia achromatica, Thielavia
albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia
fimeti, Thielavia microspora, Thielavia ovispora, Thielavia
peruviana, Thielavia setosa, Thielavia spededonium, Thielavia
subthermophila, Thielavia terrestris, Trichoderma harzianum,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma
reesei, or Trichoderma viride polypeptide.
In another aspect, the polypeptide is a Thielavia hyrcaniae
polypeptide having cellobiohydrolase activity, e.g., a polypeptide
obtained from Thielavia hyrcaniae NN045097 or Thielavia hyrcaniae
NN045178 (CGMCC 0864).
It will be understood that for the aforementioned species the
invention encompasses both the perfect and imperfect states, and
other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a
number of culture collections, such as the American Type Culture
Collection (ATCC), Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures
(CBS), and Agricultural Research Service Patent Culture Collection,
Northern Regional Research Center (NRRL).
The polypeptide may be identified and obtained from other sources
including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) using the above-mentioned probes. Techniques
for isolating microorganisms from natural habitats are well known
in the art. The polynucleotide encoding the polypeptide may then be
obtained by similarly screening a genomic or cDNA library of
another microorganism or mixed DNA sample. Once a polynucleotide
encoding a polypeptide has been detected with the probe(s), the
polynucleotide can be isolated or cloned by utilizing techniques
that are well known to those of ordinary skill in the art (see,
e.g., Sambrook et al., 1989, supra).
Polynucleotides
The present invention also relates to isolated polynucleotides
encoding a polypeptide of the present invention.
The techniques used to isolate or clone a polynucleotide encoding a
polypeptide are known in the art and include isolation from genomic
DNA, preparation from cDNA, or a combination thereof. The cloning
of the polynucleotides from such genomic DNA can be effected, e.g.,
by using the well known polymerase chain reaction (PCR) or antibody
screening of expression libraries to detect cloned DNA fragments
with shared structural features. See, e.g., Innis et al., 1990,
PCR: A Guide to Methods and Application, Academic Press, New York.
Other nucleic acid amplification procedures such as ligase chain
reaction (LCR), ligation activated transcription (LAT) and
polynucleotide-based amplification (NASBA) may be used. The
polynucleotides may be cloned from a strain of Thielavia, or
another or related organism and thus, for example, may be an
allelic or species variant of the polypeptide encoding region of
the polynucleotide.
The present invention also relates to isolated polynucleotides
comprising or consisting of polynucleotides having a degree of
sequence identity to the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3, or the cDNA sequence thereof, of at least
90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%, which encode a polypeptide having cellobiohydrolase
activity.
Modification of a polynucleotide encoding a polypeptide of the
present invention may be necessary for the synthesis of
polypeptides substantially similar to the polypeptide. The term
"substantially similar" to the polypeptide refers to non-naturally
occurring forms of the polypeptide. These polypeptides may differ
in some engineered way from the polypeptide isolated from its
native source, e.g., variants that differ in specific activity,
thermostability, pH optimum, or the like. The variant may be
constructed on the basis of the polynucleotide presented as the
mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3
or the cDNA sequence thereof, e.g., a subsequence thereof, and/or
by introduction of nucleotide substitutions that do not result in a
change in the amino acid sequence of the polypeptide, but which
correspond to the codon usage of the host organism intended for
production of the enzyme, or by introduction of nucleotide
substitutions that may give rise to a different amino acid
sequence. For a general description of nucleotide substitution,
see, e.g., Ford et al., 1991, Protein Expression and Purification
2: 95-107.
The present invention also relates to isolated polynucleotides
encoding polypeptides of the present invention, which hybridize
under high stringency conditions or very high stringency conditions
with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or
SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or
(iii) the full-length complementary strand of (i) or (ii); or
allelic variants and subsequences thereof (Sambrook et al., 1989,
supra), as defined herein.
In one aspect, the polynucleotide comprises or consists of SEQ ID
NO: 1, the mature polypeptide coding sequence of SEQ ID NO: 1, or
the sequence contained in plasmid pGEM-T-CBHII45097 which is
contained in E. coli DSM 22598, or a subsequence of SEQ ID NO: 1
that encodes a fragment of SEQ ID NO: 2 having cellobiohydrolase
activity, such as the polynucleotide of nucleotides 52 to 1595 of
SEQ ID NO: 1. In another aspect, the polynucleotide comprises or
consists of SEQ ID NO: 3, the mature polypeptide coding sequence of
SEQ ID NO: 3, or the sequence contained in plasmid
pGEM-T-CBHII45178 which is contained in E. coli DSM 22599, or a
subsequence of SEQ ID NO: 3 that encodes a fragment of SEQ ID NO: 4
having cellobiohydrolase activity, such as the polynucleotide of
nucleotides 52 to 1581 of SEQ ID NO: 3.
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs
comprising a polynucleotide of the present invention operably
linked to one or more (several) control sequences that direct the
expression of the coding sequence in a suitable host cell under
conditions compatible with the control sequences.
A polynucleotide may be manipulated in a variety of ways to provide
for expression of the polypeptide. Manipulation of the
polynucleotide prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotides utilizing recombinant DNA
methods are well known in the art.
The control sequence may be a promoter sequence, a polynucleotide
that is recognized by a host cell for expression of a
polynucleotide encoding a polypeptide of the present invention. The
promoter sequence contains transcriptional control sequences that
mediate the expression of the polypeptide. The promoter may be any
polynucleotide that shows transcriptional activity in the host cell
of choice including mutant, truncated, and hybrid promoters, and
may be obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
Examples of suitable promoters for directing the transcription of
the nucleic acid constructs of the present invention in a bacterial
host cell are the promoters obtained from the Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
alpha-amylase gene (amyL), Bacillus licheniformis penicillinase
gene (penP), Bacillus stearothermophilus maltogenic amylase gene
(amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus
subtilis xylA and xylB genes, E. coli lac operon, Streptomyces
coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene
(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:
3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc.
Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in
"Useful proteins from recombinant bacteria" in Gilbert et al.,
1980, Scientific American, 242: 74-94; and in Sambrook et al.,
1989, supra.
Examples of suitable promoters for directing the transcription of
the nucleic acid constructs of the present invention in a
filamentous fungal host cell are promoters obtained from the genes
for Aspergillus nidulans acetamidase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline
protease, Aspergillus oryzae triose phosphate isomerase, Fusarium
oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum
amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO
00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor
miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma
reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I,
Trichoderma reesei cellobiohydrolase II, Trichoderma reesei
endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma
reesei endoglucanase III, Trichoderma reesei endoglucanase IV,
Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,
Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase,
as well as the NA2-tpi promoter (a modified promoter from a gene
encoding a neutral alpha-amylase in Aspergilli in which the
untranslated leader has been replaced by an untranslated leader
from a gene encoding triose phosphate isomerase in Aspergilli;
non-limiting examples include modified promoters from the gene
encoding neutral alpha-amylase in Aspergillus niger in which the
untranslated leader has been replaced by an untranslated leader
from the gene encoding triose phosphate isomerase in Aspergillus
nidulans or Aspergillus oryzae); and mutant, truncated, and hybrid
promoters thereof.
In a yeast host, useful promoters are obtained from the genes for
Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae
galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
The control sequence may also be a suitable transcription
terminator sequence, which is recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3'-terminus of the polynucleotide encoding the polypeptide.
Any terminator that is functional in the host cell of choice may be
used in the present invention.
Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans anthranilate
synthase, Aspergillus niger glucoamylase, Aspergillus niger
alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium
oxysporum trypsin-like protease.
Preferred terminators for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
The control sequence may also be a suitable leader sequence, when
transcribed is a nontranslated region of an mRNA that is important
for translation by the host cell. The leader sequence is operably
linked to the 5'-terminus of the polynucleotide encoding the
polypeptide. Any leader sequence that is functional in the host
cell of choice may be used.
Preferred leaders for filamentous fungal host cells are obtained
from the genes for Aspergillus oryzae TAKA amylase and Aspergillus
nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes
for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a
sequence operably linked to the 3'-terminus of the polynucleotide
and, when transcribed, is recognized by the host cell as a signal
to add polyadenosine residues to transcribed mRNA. Any
polyadenylation sequence that is functional in the host cell of
choice may be used.
Preferred polyadenylation sequences for filamentous fungal host
cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
Useful polyadenylation sequences for yeast host cells are described
by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
The control sequence may also be a signal peptide coding region
that encodes a signal peptide linked to the N-terminus of a
polypeptide and directs the polypeptide into the cell's secretory
pathway. The 5'-end of the coding sequence of the polynucleotide
may inherently contain a signal peptide coding sequence naturally
linked in translation reading frame with the segment of the coding
sequence that encodes the polypeptide. Alternatively, the 5'-end of
the coding sequence may contain a signal peptide coding sequence
that is foreign to the coding sequence. The foreign signal peptide
coding sequence may be required where the coding sequence does not
naturally contain a signal peptide coding sequence. Alternatively,
the foreign signal peptide coding sequence may simply replace the
natural signal peptide coding sequence in order to enhance
secretion of the polypeptide. However, any signal peptide coding
sequence that directs the expressed polypeptide into the secretory
pathway of a host cell of choice may be used.
Effective signal peptide coding sequences for bacterial host cells
are the signal peptide coding sequences obtained from the genes for
Bacillus NCOB 11837 maltogenic amylase, Bacillus licheniformis
subtilisin, Bacillus licheniformis beta-lactamase, Bacillus
stearothermophilus alpha-amylase, Bacillus stearothermophilus
neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA.
Further signal peptides are described by Simonen and Palva, 1993,
Microbiological Reviews 57: 109-137.
Effective signal peptide coding sequences for filamentous fungal
host cells are the signal peptide coding sequences obtained from
the genes for Aspergillus niger neutral amylase, Aspergillus niger
glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens
cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa
lipase, and Rhizomucor miehei aspartic proteinase.
Useful signal peptides for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces
cerevisiae invertase. Other useful signal peptide coding sequences
are described by Romanos et al., 1992, supra.
The control sequence may also be a propeptide coding sequence that
encodes a propeptide positioned at the N-terminus of a polypeptide.
The resultant polypeptide is known as a proenzyme or propolypeptide
(or a zymogen in some cases). A propolypeptide is generally
inactive and can be converted to an active polypeptide by catalytic
or autocatalytic cleavage of the propeptide from the
propolypeptide. The propeptide coding sequence may be obtained from
the genes for Bacillus subtilis alkaline protease (aprE), Bacillus
subtilis neutral protease (nprT), Myceliophthora thermophila
laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and
Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present at
the N-terminus of a polypeptide, the propeptide sequence is
positioned next to the N-terminus of a polypeptide and the signal
peptide sequence is positioned next to the N-terminus of the
propeptide sequence.
It may also be desirable to add regulatory sequences that allow the
regulation of the expression of the polypeptide relative to the
growth of the host cell. Examples of regulatory systems are those
that cause the expression of the gene to be turned on or off in
response to a chemical or physical stimulus, including the presence
of a regulatory compound. Regulatory systems in prokaryotic systems
include the lac, tac, and trp operator systems. In yeast, the ADH2
system or GAL1 system may be used. In filamentous fungi, the
Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA
alpha-amylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used. Other examples of regulatory sequences are
those that allow for gene amplification. In eukaryotic systems,
these regulatory sequences include the dihydrofolate reductase gene
that is amplified in the presence of methotrexate, and the
metallothionein genes that are amplified with heavy metals. In
these cases, the polynucleotide encoding the polypeptide would be
operably linked with the regulatory sequence.
Expression Vectors
The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a
promoter, and transcriptional and translational stop signals. The
various nucleotide and control sequences may be joined together to
produce a recombinant expression vector that may include one or
more (several) convenient restriction sites to allow for insertion
or substitution of the polynucleotide encoding the polypeptide at
such sites. Alternatively, the polynucleotide may be expressed by
inserting the polynucleotide or a nucleic acid construct comprising
the sequence into an appropriate vector for expression. In creating
the expression vector, the coding sequence is located in the vector
so that the coding sequence is operably linked with the appropriate
control sequences for expression.
The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant
DNA procedures and can bring about expression of the
polynucleotide. The choice of the vector will typically depend on
the compatibility of the vector with the host cell into which the
vector is to be introduced. The vector may be a linear or closed
circular plasmid.
The vector may be an autonomously replicating vector, i.e., a
vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
The vector preferably contains one or more (several) selectable
markers that permit easy selection of transformed, transfected,
transduced, or the like cells. A selectable marker is a gene the
product of which provides for biocide or viral resistance,
resistance to heavy metals, prototrophy to auxotrophs, and the
like.
Examples of bacterial selectable markers are the dal genes from
Bacillus subtilis or Bacillus licheniformis, or markers that confer
antibiotic resistance such as ampicillin, chloramphenicol,
kanamycin, or tetracycline resistance. Suitable markers for yeast
host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are the amdS and pyrG genes of Aspergillus nidulans or
Aspergillus oryzae and the bar gene of Streptomyces
hygroscopicus.
The vector preferably contains an element(s) that permits
integration of the vector into the host cell's genome or autonomous
replication of the vector in the cell independent of the
genome.
For integration into the host cell genome, the vector may rely on
the polynucleotide's sequence encoding the polypeptide or any other
element of the vector for integration into the genome by homologous
or non-homologous recombination. Alternatively, the vector may
contain additional polynucleotides for directing integration by
homologous recombination into the genome of the host cell at a
precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should contain a sufficient number of nucleic acids, such
as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to
10,000 base pairs, which have a high degree of sequence identity to
the corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host cell. Furthermore, the integrational elements may be
non-encoding or encoding polynucleotides. On the other hand, the
vector may be integrated into the genome of the host cell by
non-homologous recombination.
For autonomous replication, the vector may further comprise an
origin of replication enabling the vector to replicate autonomously
in the host cell in question. The origin of replication may be any
plasmid replicator mediating autonomous replication that functions
in a cell. The term "origin of replication" or "plasmid replicator"
means a polynucleotide that enables a plasmid or vector to
replicate in vivo.
Examples of bacterial origins of replication are the origins of
replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM.beta.1 permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are
the 2 micron origin of replication, ARS1, ARS4, the combination of
ARS1 and CEN3, and the combination of ARS4 and CEN6.
Examples of origins of replication useful in a filamentous fungal
cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen
et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883).
Isolation of the AMA1 gene and construction of plasmids or vectors
comprising the gene can be accomplished according to the methods
disclosed in WO 00/24883.
More than one copy of a polynucleotide of the present invention may
be inserted into a host cell to increase production of a
polypeptide. An increase in the copy number of the polynucleotide
can be obtained by integrating at least one additional copy of the
sequence into the host cell genome or by including an amplifiable
selectable marker gene with the polynucleotide where cells
containing amplified copies of the selectable marker gene, and
thereby additional copies of the polynucleotide, can be selected
for by cultivating the cells in the presence of the appropriate
selectable agent.
The procedures used to ligate the elements described above to
construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
The present invention also relates to recombinant host cells,
comprising a polynucleotide of the present invention operably
linked to one or more (several) control sequences that direct the
production of a polypeptide of the present invention. A construct
or vector comprising a polynucleotide is introduced into a host
cell so that the construct or vector is maintained as a chromosomal
integrant or as a self-replicating extra-chromosomal vector as
described earlier. The term "host cell" encompasses any progeny of
a parent cell that is not identical to the parent cell due to
mutations that occur during replication. The choice of a host cell
will to a large extent depend upon the gene encoding the
polypeptide and its source.
The host cell may be any cell useful in the recombinant production
of a polypeptide of the present invention, e.g., a prokaryote or a
eukaryote.
The prokaryotic host cell may be any gram-positive or gram-negative
bacterium. Gram-positive bacteria include, but not limited to,
Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,
Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and
Streptomyces. Gram-negative bacteria include, but not limited to,
Campylobacter, E. coli, Flavobacterium, Fusobacterium,
Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and
Ureaplasma.
The bacterial host cell may be any Bacillus cell including, but not
limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens,
Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus
coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus,
Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,
Bacillus stearothermophilus, Bacillus subtilis, and Bacillus
thuringiensis cells.
The bacterial host cell may also be any Streptococcus cell
including, but not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell
including, but not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
The introduction of DNA into a Bacillus cell may, for instance, be
effected by protoplast transformation (see, e.g., Chang and Cohen,
1979, Mol. Gen. Genet. 168: 111-115), by using competent cells
(see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or
Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by
electroporation (see, e.g., Shigekawa and Dower, 1988,
Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler
and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction
of DNA into an E. coli cell may, for instance, be effected by
protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.
166: 557-580) or electroporation (see, e.g., Dower et al., 1988,
Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a
Streptomyces cell may, for instance, be effected by protoplast
transformation and electroporation (see, e.g., Gong et al., 2004,
Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g.,
Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by
transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci.
USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell
may, for instance, be effected by electroporation (see, e.g., Choi
et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation
(see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:
51-57). The introduction of DNA into a Streptococcus cell may, for
instance, be effected by natural competence (see, e.g., Perry and
Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast
transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:
189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.
Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,
Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method
known in the art for introducing DNA into a host cell can be
used.
The host cell may also be a eukaryote, such as a mammalian, insect,
plant, or fungal cell.
The host cell may be a fungal cell. "Fungi" as used herein includes
the phyla Ascomycota, Basidiomycota, Chytridiomycota, and
Zygomycota (as defined by Hawksworth et al., In, Ainsworth and
Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB
International, University Press, Cambridge, UK) as well as the
Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and
all mitosporic fungi (Hawksworth et al., 1995, supra).
The fungal host cell may be a yeast cell. "Yeast" as used herein
includes ascosporogenous yeast (Endomycetales), basidiosporogenous
yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).
Since the classification of yeast may change in the future, for the
purposes of this invention, yeast shall be defined as described in
Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M.,
and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series
No. 9, 1980).
The yeast host cell may be a Candida, Hansenula, Kluyveromyces,
Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such
as a Kluyveromyces lactis, Saccharomyces carlsbergensis,
Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces
douglasii, Saccharomyces kluyveri, Saccharomyces norbensis,
Saccharomyces oviformis, or Yarrowia lipolytica cell.
The fungal host cell may be a filamentous fungal cell. "Filamentous
fungi" include all filamentous forms of the subdivision Eumycota
and Oomycota (as defined by Hawksworth et al., 1995, supra). The
filamentous fungi are generally characterized by a mycelial wall
composed of chitin, cellulose, glucan, chitosan, mannan, and other
complex polysaccharides. Vegetative growth is by hyphal elongation
and carbon catabolism is obligately aerobic. In contrast,
vegetative growth by yeasts such as Saccharomyces cerevisiae is by
budding of a unicellular thallus and carbon catabolism may be
fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus,
Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,
Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,
Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,
Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces,
Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia,
Tolypocladium, Trametes, or Trichoderma cell.
For example, the filamentous fungal host cell may be an Aspergillus
awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus
japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus
oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis
caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta,
Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis
subvermispora, Chrysosporium inops, Chrysosporium keratinophilum,
Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium
pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum,
Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus,
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,
Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium
sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium
venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
Fungal cells may be transformed by a process involving protoplast
formation, transformation of the protoplasts, and regeneration of
the cell wall in a manner known per se. Suitable procedures for
transformation of Aspergillus and Trichoderma host cells are
described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad.
Sci. USA 81: 1470-1474. Suitable methods for transforming Fusarium
species are described by Malardier et al., 1989, Gene 78: 147-156,
and WO 96/00787. Yeast may be transformed using the procedures
described by Becker and Guarente, In Abelson, J. N. and Simon, M.
I., editors, Guide to Yeast Genetics and Molecular Biology, Methods
in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New
York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al.,
1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Methods of Production
The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
cell, which in its wild-type form produces the polypeptide, under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide. In a preferred aspect, the cell is of
the genus Thielavia. In a more preferred aspect, the cell is
Thielavia hyrcaniae. In a most preferred aspect, the cell is
Thielavia hyrcaniae NN045097. In another most preferred aspect, the
cell is Thielavia hyrcaniae NN045178 (CGMCC 0864).
The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
recombinant host cell of the present invention under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide.
The host cells are cultivated in a nutrient medium suitable for
production of the polypeptide using methods well known in the art.
For example, the cell may be cultivated by shake flask cultivation,
and small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted, it can be recovered from cell
lysates.
The polypeptide may be detected using methods known in the art that
are specific for the polypeptides. These detection methods may
include use of specific antibodies, formation of an enzyme product,
or disappearance of an enzyme substrate. For example, an enzyme
assay may be used to determine the activity of the polypeptide.
The polypeptide may be recovered using methods known in the art.
For example, the polypeptide may be recovered from the nutrient
medium by conventional procedures including, but not limited to,
centrifugation, filtration, extraction, spray-drying, evaporation,
or precipitation.
The polypeptide may be purified by a variety of procedures known in
the art including, but not limited to, chromatography (e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size
exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989) to obtain substantially pure polypeptides.
In an alternative aspect, the polypeptide is not recovered, but
rather a host cell of the present invention expressing the
polypeptide is used as a source of the polypeptide.
Plants
The present invention also relates to isolated plants, e.g., a
transgenic plant, plant part, or plant cell, comprising an isolated
polynucleotide of the present invention so as to express and
produce the polypeptide in recoverable quantities. The polypeptide
may be recovered from the plant or plant part. Alternatively, the
plant or plant part containing the polypeptide may be used as such
for improving the quality of a food or feed, e.g., improving
nutritional value, palatability, and rheological properties, or to
destroy an antinutritive factor.
The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
Examples of dicot plants are tobacco, legumes, such as lupins,
potato, sugar beet, pea, bean and soybean, and cruciferous plants
(family Brassicaceae), such as cauliflower, rape seed, and the
closely related model organism Arabidopsis thaliana.
Examples of plant parts are stem, callus, leaves, root, fruits,
seeds, and tubers as well as the individual tissues comprising
these parts, e.g., epidermis, mesophyll, parenchyme, vascular
tissues, meristems. Specific plant cell compartments, such as
chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilization of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seeds coats.
Also included within the scope of the present invention are the
progeny of such plants, plant parts, and plant cells.
The transgenic plant or plant cell expressing a polypeptide may be
constructed in accordance with methods known in the art. In short,
the plant or plant cell is constructed by incorporating one or more
(several) expression constructs encoding a polypeptide into the
plant host genome or chloroplast genome and propagating the
resulting modified plant or plant cell into a transgenic plant or
plant cell.
The expression construct is conveniently a nucleic acid construct
that comprises a polynucleotide encoding a polypeptide operably
linked with appropriate regulatory sequences required for
expression of the polynucleotide in the plant or plant part of
choice. Furthermore, the expression construct may comprise a
selectable marker useful for identifying host cells into which the
expression construct has been integrated and DNA sequences
necessary for introduction of the construct into the plant in
question (the latter depends on the DNA introduction method to be
used).
The choice of regulatory sequences, such as promoter and terminator
sequences and optionally signal or transit sequences, is
determined, for example, on the basis of when, where, and how the
polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide may be constitutive
or inducible, or may be developmental, stage or tissue specific,
and the gene product may be targeted to a specific tissue or plant
part such as seeds or leaves. Regulatory sequences are, for
example, described by Tague et al., 1988, Plant Physiology 86:
506.
For constitutive expression, the 35S-CaMV, the maize ubiquitin 1,
and the rice actin 1 promoter may be used (Franck et al., 1980,
Cell 21: 285-294; Christensen et al., 1992, Plant Mol. Biol. 18:
675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from
metabolic sink tissues such as meristems (Ito et al., 1994, Plant
Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter
from the legumin B4 and the unknown seed protein gene from Vicia
faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a
promoter from a seed oil body protein (Chen et al., 1998, Plant
Cell Physiol. 39: 935-941), the storage protein napA promoter from
Brassica napus, or any other seed specific promoter known in the
art, e.g., as described in WO 91/14772. Furthermore, the promoter
may be a leaf specific promoter such as the rbcs promoter from rice
or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter
from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or
a wound inducible promoter such as the potato pin2 promoter (Xu et
al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter
may inducible by abiotic treatments such as temperature, drought,
or alterations in salinity or induced by exogenously applied
substances that activate the promoter, e.g., ethanol, oestrogens,
plant hormones such as ethylene, abscisic acid, and gibberellic
acid, and heavy metals.
A promoter enhancer element may also be used to achieve higher
expression of a polypeptide in the plant. For instance, the
promoter enhancer element may be an intron that is placed between
the promoter and the polynucleotide encoding a polypeptide. For
instance, Xu et al., 1993, supra, disclose the use of the first
intron of the rice actin 1 gene to enhance expression.
The selectable marker gene and any other parts of the expression
construct may be chosen from those available in the art.
The nucleic acid construct is incorporated into the plant genome
according to conventional techniques known in the art, including
Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
Presently, Agrobacterium tumefaciens-mediated gene transfer is the
method of choice for generating transgenic dicots (for a review,
see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and
can also be used for transforming monocots, although other
transformation methods are often used for these plants. Presently,
the method of choice for generating transgenic monocots is particle
bombardment (microscopic gold or tungsten particles coated with the
transforming DNA) of embryonic calli or developing embryos
(Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin.
Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10:
667-674). An alternative method for transformation of monocots is
based on protoplast transformation as described by Omirulleh et
al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformation
methods for use in accordance with the present disclosure include
those described in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of
which are herein incorporated by reference in their entirety).
Following transformation, the transformants having incorporated the
expression construct are selected and regenerated into whole plants
according to methods well known in the art. Often the
transformation procedure is designed for the selective elimination
of selection genes either during regeneration or in the following
generations by using, for example, co-transformation with two
separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
In addition to direct transformation of a particular plant genotype
with a construct prepared according to the present invention,
transgenic plants may be made by crossing a plant having the
construct to a second plant lacking the construct. For example, a
construct encoding a polypeptide can be introduced into a
particular plant variety by crossing, without the need for ever
directly transforming a plant of that given variety. Therefore, the
present invention encompasses not only a plant directly regenerated
from cells which have been transformed in accordance with the
present invention, but also the progeny of such plants. As used
herein, progeny may refer to the offspring of any generation of a
parent plant prepared in accordance with the present invention.
Such progeny may include a DNA construct prepared in accordance
with the present invention, or a portion of a DNA construct
prepared in accordance with the present invention. Crossing results
in the introduction of a transgene into a plant line by cross
pollinating a starting line with a donor plant line. Non-limiting
examples of such steps are further articulated in U.S. Pat. No.
7,151,204.
Plants may be generated through a process of backcross conversion.
For example, plants include plants referred to as a backcross
converted genotype, line, inbred, or hybrid.
Genetic markers may be used to assist in the introgression of one
or more transgenes of the invention from one genetic background
into another. Marker assisted selection offers advantages relative
to conventional breeding in that it can be used to avoid errors
caused by phenotypic variations. Further, genetic markers may
provide data regarding the relative degree of elite germplasm in
the individual progeny of a particular cross. For example, when a
plant with a desired trait which otherwise has a non-agronomically
desirable genetic background is crossed to an elite parent, genetic
markers may be used to select progeny which not only possess the
trait of interest, but also have a relatively large proportion of
the desired germplasm. In this way, the number of generations
required to introgress one or more traits into a particular genetic
background is minimized.
The present invention also relates to methods of producing a
polypeptide of the present invention comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding the polypeptide under conditions conducive for production
of the polypeptide; and (b) recovering the polypeptide.
Removal or Reduction of Cellobiohydrolase Activity
The present invention also relates to methods of producing a mutant
of a parent cell, which comprises disrupting or deleting a
polynucleotide, or a portion thereof, encoding a polypeptide of the
present invention, which results in the mutant cell producing less
of the polypeptide than the parent cell when cultivated under the
same conditions.
The mutant cell may be constructed by reducing or eliminating
expression of the polynucleotide using methods well known in the
art, for example, insertions, disruptions, replacements, or
deletions. In a preferred aspect, the polynucleotide is
inactivated. The polynucleotide to be modified or inactivated may
be, for example, the coding region or a part thereof essential for
activity, or a regulatory element required for the expression of
the coding region. An example of such a regulatory or control
sequence may be a promoter sequence or a functional part thereof,
i.e., a part that is sufficient for affecting expression of the
polynucleotide. Other control sequences for possible modification
include, but are not limited to, a leader, polyadenylation
sequence, propeptide sequence, signal peptide sequence,
transcription terminator, and transcriptional activator.
Modification or inactivation of the polynucleotide may be performed
by subjecting the parent cell to mutagenesis and selecting for
mutant cells in which expression of the polynucleotide has been
reduced or eliminated. The mutagenesis, which may be specific or
random, may be performed, for example, by use of a suitable
physical or chemical mutagenizing agent, by use of a suitable
oligonucleotide, or by subjecting the DNA sequence to PCR generated
mutagenesis. Furthermore, the mutagenesis may be performed by use
of any combination of these mutagenizing agents.
Examples of a physical or chemical mutagenizing agent suitable for
the present purpose include ultraviolet (UV) irradiation,
hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG),
O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate
(EMS), sodium bisulphite, formic acid, and nucleotide
analogues.
When such agents are used, the mutagenesis is typically performed
by incubating the parent cell to be mutagenized in the presence of
the mutagenizing agent of choice under suitable conditions, and
screening and/or selecting for mutant cells exhibiting reduced or
no expression of the gene.
Modification or inactivation of the polynucleotide may be
accomplished by introduction, substitution, or removal of one or
more (several) nucleotides in the gene or a regulatory element
required for the transcription or translation thereof. For example,
nucleotides may be inserted or removed so as to result in the
introduction of a stop codon, the removal of the start codon, or a
change in the open reading frame. Such modification or inactivation
may be accomplished by site-directed mutagenesis or PCR generated
mutagenesis in accordance with methods known in the art. Although,
in principle, the modification may be performed in vivo, i.e.,
directly on the cell expressing the polynucleotide to be modified,
it is preferred that the modification be performed in vitro as
exemplified below.
An example of a convenient way to eliminate or reduce expression of
a polynucleotide is based on techniques of gene replacement, gene
deletion, or gene disruption. For example, in the gene disruption
method, a nucleic acid sequence corresponding to the endogenous
polynucleotide is mutagenized in vitro to produce a defective
nucleic acid sequence that is then transformed into the parent cell
to produce a defective gene. By homologous recombination, the
defective nucleic acid sequence replaces the endogenous
polynucleotide. It may be desirable that the defective
polynucleotide also encodes a marker that may be used for selection
of transformants in which the polynucleotide has been modified or
destroyed. In a particularly preferred aspect, the polynucleotide
is disrupted with a selectable marker such as those described
herein.
The present invention also relates to methods of inhibiting the
expression of a polypeptide having cellobiohydrolase activity in a
cell, comprising administering to the cell or expressing in the
cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA
comprises a subsequence of a polynucleotide of the present
invention. In a preferred aspect, the dsRNA is about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in
length.
The dsRNA is preferably a small interfering RNA (siRNA) or a micro
RNA (miRNA). In a preferred aspect, the dsRNA is small interfering
RNA (siRNAs) for inhibiting transcription. In another preferred
aspect, the dsRNA is micro RNA (miRNAs) for inhibiting
translation.
The present invention also relates to such double-stranded RNA
(dsRNA) molecules, comprising a portion of the mature polypeptide
coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 for inhibiting
expression of the polypeptide in a cell. While the present
invention is not limited by any particular mechanism of action, the
dsRNA can enter a cell and cause the degradation of a
single-stranded RNA (ssRNA) of similar or identical sequences,
including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA
from the homologous gene is selectively degraded by a process
called RNA interference (RNAi).
The dsRNAs of the present invention can be used in gene-silencing.
In one aspect, the invention provides methods to selectively
degrade RNA using a dsRNAi of the present invention. The process
may be practiced in vitro, ex vivo or in vivo. In one aspect, the
dsRNA molecules can be used to generate a loss-of-function mutation
in a cell, an organ or an animal. Methods for making and using
dsRNA molecules to selectively degrade RNA are well known in the
art; see, for example, U.S. Pat. Nos. 6,489,127; 6,506,559;
6,511,824; and 6,515,109.
The present invention further relates to a mutant cell of a parent
cell that comprises a disruption or deletion of a polynucleotide
encoding the polypeptide or a control sequence thereof or a
silenced gene encoding the polypeptide, which results in the mutant
cell producing less of the polypeptide or no polypeptide compared
to the parent cell.
The polypeptide-deficient mutant cells are particularly useful as
host cells for the expression of native and heterologous
polypeptides. Therefore, the present invention further relates to
methods of producing a native or heterologous polypeptide,
comprising: (a) cultivating the mutant cell under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide. The term "heterologous polypeptides" means
polypeptides that are not native to the host cell, e.g., a variant
of a native protein. The host cell may comprise more than one copy
of a polynucleotide encoding the native or heterologous
polypeptide.
The methods used for cultivation and purification of the product of
interest may be performed by methods known in the art.
The methods of the present invention for producing an essentially
cellobiohydrolase-free product is of particular interest in the
production of eukaryotic polypeptides, in particular fungal
proteins such as enzymes. The cellobiohydrolase-deficient cells may
also be used to express heterologous proteins of pharmaceutical
interest such as hormones, growth factors, receptors, and the like.
The term "eukaryotic polypeptides" includes not only native
polypeptides, but also those polypeptides, e.g., enzymes, which
have been modified by amino acid substitutions, deletions or
additions, or other such modifications to enhance activity,
thermostability, pH tolerance and the like.
In a further aspect, the present invention relates to a protein
product essentially free from cellobiohydrolase activity that is
produced by a method of the present invention.
Compositions
The present invention also relates to compositions comprising a
polypeptide of the present invention. Preferably, the compositions
are enriched in such a polypeptide. The term "enriched" indicates
that the cellobiohydrolase activity of the composition has been
increased, e.g., with an enrichment factor of at least 1.1.
The composition may comprise a polypeptide of the present invention
as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,
esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase,
laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,
peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The additional enzyme(s) may be produced, for example, by a
microorganism belonging to the genus Aspergillus, preferably
Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,
Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, or Aspergillus oryzae; Fusarium, preferably
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,
Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum,
Fusarium trichothecioides, or Fusarium venenatum; Humicola,
preferably Humicola insolens or Humicola lanuginosa; or
Trichoderma, preferably Trichoderma harzianum, Trichoderma
koningii, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma viride.
The polypeptide compositions may be prepared in accordance with
methods known in the art and may be in the form of a liquid or a
dry composition. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art.
Examples are given below of preferred uses of the polypeptide
compositions of the invention. The dosage of the polypeptide
composition of the invention and other conditions under which the
composition is used may be determined on the basis of methods known
in the art.
Uses
The present invention is also directed to the following methods for
using the polypeptides having cellobiohydrolase activity, or
compositions thereof.
The present invention also relates to methods for degrading or
converting a cellulosic material, comprising: treating the
cellulosic material with an enzyme composition in the presence of a
polypeptide having cellobiohydrolase activity of the present
invention. In a preferred aspect, the method further comprises
recovering the degraded or converted cellulosic material.
The present invention also relates to methods of producing a
fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a
polypeptide having cellobiohydrolase activity of the present
invention; (b) fermenting the saccharified cellulosic material with
one or more (several) fermenting microorganisms to produce the
fermentation product; and (c) recovering the fermentation product
from the fermentation.
The present invention also relates to methods of fermenting a
cellulosic material, comprising: fermenting the cellulosic material
with one or more (several) fermenting microorganisms, wherein the
cellulosic material is saccharified with an enzyme composition in
the presence of a polypeptide having cellobiohydrolase activity of
the present invention. In a preferred aspect, the fermenting of the
cellulosic material produces a fermentation product. In another
preferred aspect, the method further comprises recovering the
fermentation product from the fermentation.
The methods of the present invention can be used to saccharify a
cellulosic material to fermentable sugars and convert the
fermentable sugars to many useful substances, e.g., fuel, potable
ethanol, and/or fermentation products (e.g., acids, alcohols,
ketones, gases, and the like). The production of a desired
fermentation product from cellulosic material typically involves
pretreatment, enzymatic hydrolysis (saccharification), and
fermentation.
The processing of cellulosic material according to the present
invention can be accomplished using processes conventional in the
art. Moreover, the methods of the present invention can be
implemented using any conventional biomass processing apparatus
configured to operate in accordance with the invention.
Hydrolysis (saccharification) and fermentation, separate or
simultaneous, include, but are not limited to, separate hydrolysis
and fermentation (SHF); simultaneous saccharification and
fermentation (SSF); simultaneous saccharification and
cofermentation (SSCF); hybrid hydrolysis and fermentation (HHF);
separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis
and co-fermentation (HHCF); and direct microbial conversion (DMC).
SHF uses separate process steps to first enzymatically hydrolyze
cellulosic material to fermentable sugars, e.g., glucose,
cellobiose, cellotriose, and pentose sugars, and then ferment the
fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of
cellulosic material and the fermentation of sugars to ethanol are
combined in one step (Philippidis, G. P., 1996, Cellulose
bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
D.C., 179-212). SSCF involves the cofermentation of multiple sugars
(Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the
environment: A strategic perspective on the U.S. Department of
Energy's research and development activities for bioethanol,
Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysis
step, and in addition a simultaneous saccharification and
hydrolysis step, which can be carried out in the same reactor. The
steps in an HHF process can be carried out at different
temperatures, i.e., high temperature enzymatic saccharification
followed by SSF at a lower temperature that the fermentation strain
can tolerate. DMC combines all three processes (enzyme production,
hydrolysis, and fermentation) in one or more (several) steps where
the same organism is used to produce the enzymes for conversion of
the cellulosic material to fermentable sugars and to convert the
fermentable sugars into a final product (Lynd, L. R., Weimer, P.
J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose
utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol.
Reviews 66: 506-577). It is understood herein that any method known
in the art comprising pretreatment, enzymatic hydrolysis
(saccharification), fermentation, or a combination thereof, can be
used in the practicing the methods of the present invention.
A conventional apparatus can include a fed-batch stirred reactor, a
batch stirred reactor, a continuous flow stirred reactor with
ultrafiltration, and/or a continuous plug-flow column reactor
(Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella
Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch
reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology
25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of
the enzymatic hydrolysis of cellulose: 1. A mathematical model for
a batch reactor process, Enz. Microb. Technol. 7: 346-352), an
attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion
of waste cellulose by using an attrition bioreactor, Biotechnol.
Bioeng. 25: 53-65), or a reactor with intensive stirring induced by
an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P.,
Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement
of enzymatic cellulose hydrolysis using a novel type of bioreactor
with intensive stirring induced by electromagnetic field, Appl.
Biochem. Biotechnol. 56: 141-153). Additional reactor types
include: fluidized bed, upflow blanket, immobilized, and extruder
type reactors for hydrolysis and/or fermentation.
Pretreatment. In practicing the methods of the present invention,
any pretreatment process known in the art can be used to disrupt
plant cell wall components of cellulosic material (Chandra et al.,
2007, Substrate pretreatment: The key to effective enzymatic
hydrolysis of lignocellulosics? Adv. Biochem. Engin./Biotechnol.
108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic
materials for efficient bioethanol production, Adv. Biochem.
Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,
Pretreatments to enhance the digestibility of lignocellulosic
biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005,
Features of promising technologies for pretreatment of
lignocellulosic biomass, Bioresource Technol. 96: 673-686;
Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes
to improve ethanol and biogas production: A review, Int. J. of Mol.
Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to
unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and
Biorefining-Biofpr. 2: 26-40).
The cellulosic material can also be subjected to particle size
reduction, pre-soaking, wetting, washing, or conditioning prior to
pretreatment using methods known in the art.
Conventional pretreatments include, but are not limited to, steam
pretreatment (with or without explosion), dilute acid pretreatment,
hot water pretreatment, alkaline pretreatment, lime pretreatment,
wet oxidation, wet explosion, ammonia fiber explosion, organosolv
pretreatment, and biological pretreatment. Additional pretreatments
include ammonia percolation, ultrasound, electroporation,
microwave, supercritical CO.sub.2, supercritical H.sub.2O, ozone,
and gamma irradiation pretreatments.
The cellulosic material can be pretreated before hydrolysis and/or
fermentation. Pretreatment is preferably performed prior to the
hydrolysis. Alternatively, the pretreatment can be carried out
simultaneously with enzyme hydrolysis to release fermentable
sugars, such as glucose, xylose, and/or cellobiose. In most cases
the pretreatment step itself results in some conversion of biomass
to fermentable sugars (even in absence of enzymes).
Steam Pretreatment: In steam pretreatment, cellulosic material is
heated to disrupt the plant cell wall components, including lignin,
hemicellulose, and cellulose to make the cellulose and other
fractions, e.g., hemicellulose, accessible to enzymes. Cellulosic
material is passed to or through a reaction vessel where steam is
injected to increase the temperature to the required temperature
and pressure and is retained therein for the desired reaction time.
Steam pretreatment is preferably done at 140-230.degree. C., more
preferably 160-200.degree. C., and most preferably 170-190.degree.
C., where the optimal temperature range depends on any addition of
a chemical catalyst. Residence time for the steam pretreatment is
preferably 1-15 minutes, more preferably 3-12 minutes, and most
preferably 4-10 minutes, where the optimal residence time depends
on temperature range and any addition of a chemical catalyst. Steam
pretreatment allows for relatively high solids loadings, so that
cellulosic material is generally only moist during the
pretreatment. The steam pretreatment is often combined with an
explosive discharge of the material after the pretreatment, which
is known as steam explosion, that is, rapid flashing to atmospheric
pressure and turbulent flow of the material to increase the
accessible surface area by fragmentation (Duff and Murray, 1996,
Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.
Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.
20020164730). During steam pretreatment, hemicellulose acetyl
groups are cleaved and the resulting acid autocatalyzes partial
hydrolysis of the hemicellulose to monosaccharides and
oligosaccharides. Lignin is removed to only a limited extent.
A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically 0.3 to 3%
w/w) is often added prior to steam pretreatment, which decreases
the time and temperature, increases the recovery, and improves
enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem.
Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem.
Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme Microb.
Technol. 39: 756-762).
Chemical Pretreatment: The term "chemical treatment" refers to any
chemical pretreatment that promotes the separation and/or release
of cellulose, hemicellulose, and/or lignin. Examples of suitable
chemical pretreatment processes include, for example, dilute acid
pretreatment, lime pretreatment, wet oxidation, ammonia
fiber/freeze explosion (AFEX), ammonia percolation (APR), and
organosolv pretreatments.
In dilute acid pretreatment, cellulosic material is mixed with
dilute acid, typically H.sub.2SO.sub.4, and water to form a slurry,
heated by steam to the desired temperature, and after a residence
time flashed to atmospheric pressure. The dilute acid pretreatment
can be performed with a number of reactor designs, e.g., plug-flow
reactors, counter-current reactors, or continuous counter-current
shrinking bed reactors (Duff and Murray, 1996, supra; Schell et
al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv.
Biochem. Eng. Biotechnol. 65: 93-115).
Several methods of pretreatment under alkaline conditions can also
be used. These alkaline pretreatments include, but are not limited
to, lime pretreatment, wet oxidation, ammonia percolation (APR),
and ammonia fiber/freeze explosion (AFEX).
Lime pretreatment is performed with calcium carbonate, sodium
hydroxide, or ammonia at low temperatures of 85-150.degree. C. and
residence times from 1 hour to several days (Wyman et al., 2005,
Bioresource Technol. 96: 1959-1966; Mosier et al., 2005,
Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/11899,
WO 2006/11900, and WO 2006/110901 disclose pretreatment methods
using ammonia.
Wet oxidation is a thermal pretreatment performed typically at
180-200.degree. C. for 5-15 minutes with addition of an oxidative
agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt
and Thomsen, 1998, Bioresource Technol. 64: 139-151; Palonen et
al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al.,
2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J.
Chem. Technol. Biotechnol. 81: 1669-1677). The pretreatment is
performed at preferably 1-40% dry matter, more preferably 2-30% dry
matter, and most preferably 5-20% dry matter, and often the initial
pH is increased by the addition of alkali such as sodium
carbonate.
A modification of the wet oxidation pretreatment method, known as
wet explosion (combination of wet oxidation and steam explosion),
can handle dry matter up to 30%. In wet explosion, the oxidizing
agent is introduced during pretreatment after a certain residence
time. The pretreatment is then ended by flashing to atmospheric
pressure (WO 2006/032282).
Ammonia fiber explosion (AFEX) involves treating cellulosic
material with liquid or gaseous ammonia at moderate temperatures
such as 90-100.degree. C. and high pressure such as 17-20 bar for
5-10 minutes, where the dry matter content can be as high as 60%
(Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35;
Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh
et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri
et al., 2005, Bioresource Technol. 96: 2014-2018). AFEX
pretreatment results in the depolymerization of cellulose and
partial hydrolysis of hemicellulose. Lignin-carbohydrate complexes
are cleaved.
Organosolv pretreatment delignifies cellulosic material by
extraction using aqueous ethanol (40-60% ethanol) at
160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol.
Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94:
851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121:
219-230). Sulphuric acid is usually added as a catalyst. In
organosolv pretreatment, the majority of hemicellulose is
removed.
Other examples of suitable pretreatment methods are described by
Schell et al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108,
p. 69-85, and Mosier et al., 2005, Bioresource Technology 96:
673-686, and U.S. Published Application 2002/0164730.
In one aspect, the chemical pretreatment is preferably carried out
as an acid treatment, and more preferably as a continuous dilute
and/or mild acid treatment. The acid is typically sulfuric acid,
but other acids can also be used, such as acetic acid, citric acid,
nitric acid, phosphoric acid, tartaric acid, succinic acid,
hydrogen chloride, or mixtures thereof. Mild acid treatment is
conducted in the pH range of preferably 1-5, more preferably 1-4,
and most preferably 1-3. In one aspect, the acid concentration is
in the range from preferably 0.01 to 20 wt % acid, more preferably
0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt % acid, and
most preferably 0.2 to 2.0 wt % acid. The acid is contacted with
cellulosic material and held at a temperature in the range of
preferably 160-220.degree. C., and more preferably 165-195.degree.
C., for periods ranging from seconds to minutes to, e.g., 1 second
to 60 minutes.
In another aspect, pretreatment is carried out as an ammonia fiber
explosion step (AFEX pretreatment step).
In another aspect, pretreatment takes place in an aqueous slurry.
In preferred aspects, cellulosic material is present during
pretreatment in amounts preferably between 10-80 wt %, more
preferably between 20-70 wt %, and most preferably between 30-60 wt
%, such as around 50 wt %. The pretreated cellulosic material can
be unwashed or washed using any method known in the art, e.g.,
washed with water.
Mechanical Pretreatment: The term "mechanical pretreatment" refers
to various types of grinding or milling (e.g., dry milling, wet
milling, or vibratory ball milling).
Physical Pretreatment: The term "physical pretreatment" refers to
any pretreatment that promotes the separation and/or release of
cellulose, hemicellulose, and/or lignin from cellulosic material.
For example, physical pretreatment can involve irradiation (e.g.,
microwave irradiation), steaming/steam explosion, hydrothermolysis,
and combinations thereof.
Physical pretreatment can involve high pressure and/or high
temperature (steam explosion). In one aspect, high pressure means
pressure in the range of preferably about 300 to about 600 psi,
more preferably about 350 to about 550 psi, and most preferably
about 400 to about 500 psi, such as around 450 psi. In another
aspect, high temperature means temperatures in the range of about
100 to about 300.degree. C., preferably about 140 to about
235.degree. C. In a preferred aspect, mechanical pretreatment is
performed in a batch-process, steam gun hydrolyzer system that uses
high pressure and high temperature as defined above, e.g., a Sunds
Hydrolyzer available from Sunds Defibrator AB, Sweden.
Combined Physical and Chemical Pretreatment: Cellulosic material
can be pretreated both physically and chemically. For instance, the
pretreatment step can involve dilute or mild acid treatment and
high temperature and/or pressure treatment. The physical and
chemical pretreatments can be carried out sequentially or
simultaneously, as desired. A mechanical pretreatment can also be
included.
Accordingly, in a preferred aspect, cellulosic material is
subjected to mechanical, chemical, or physical pretreatment, or any
combination thereof, to promote the separation and/or release of
cellulose, hemicellulose, and/or lignin.
Biological Pretreatment: The term "biological pretreatment" refers
to any biological pretreatment that promotes the separation and/or
release of cellulose, hemicellulose, and/or lignin from cellulosic
material. Biological pretreatment techniques can involve applying
lignin-solubilizing microorganisms (see, for example, Hsu, T.-A.,
1996, Pretreatment of biomass, in Handbook on Bioethanol:
Production and Utilization, Wyman, C. E., ed., Taylor &
Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993,
Physicochemical and biological treatments for enzymatic/microbial
conversion of cellulosic biomass, Adv. Appl. Microbiol. 39:
295-333; McMillan, J. D., 1994, Pretreating lignocellulosic
biomass: a review, in Enzymatic Conversion of Biomass for Fuels
Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds.,
ACS Symposium Series 566, American Chemical Society, Washington,
D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,
1999, Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson and
Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates
for ethanol production, Enz. Microb. Tech. 18: 312-331; and
Vallander and Eriksson, 1990, Production of ethanol from
lignocellulosic materials: State of the art, Adv. Biochem.
Eng./Biotechnol. 42: 63-95).
Saccharification. In the hydrolysis step, also known as
saccharification, the cellulosic material, e.g., pretreated, is
hydrolyzed to break down cellulose and alternatively also
hemicellulose to fermentable sugars, such as glucose, cellobiose,
xylose, xylulose, arabinose, mannose, galactose, and/or soluble
oligosaccharides. The hydrolysis is performed enzymatically by an
enzyme composition in the presence of a polypeptide having
cellobiohydrolase activity of the present invention. The
composition can further comprise one or more (several)
hemicellulolytic or xylan degrading enzymes. The enzyme and protein
components of the compositions can be added sequentially.
Enzymatic hydrolysis is preferably carried out in a suitable
aqueous environment under conditions that can be readily determined
by one skilled in the art. In a preferred aspect, hydrolysis is
performed under conditions suitable for the activity of the
enzyme(s), i.e., optimal for the enzyme(s). The hydrolysis can be
carried out as a fed batch or continuous process where the
pretreated cellulosic material (substrate) is fed gradually to, for
example, an enzyme containing hydrolysis solution.
The saccharification is generally performed in stirred-tank
reactors or fermentors under controlled pH, temperature, and mixing
conditions. Suitable process time, temperature and pH conditions
can readily be determined by one skilled in the art. For example,
the saccharification can last up to 200 hours, but is typically
performed for preferably about 12 to about 96 hours, more
preferably about 16 to about 72 hours, and most preferably about 24
to about 48 hours. The temperature is in the range of preferably
about 25.degree. C. to about 70.degree. C., more preferably about
30.degree. C. to about 65.degree. C., and more preferably about
40.degree. C. to 60.degree. C., in particular about 50.degree. C.
The pH is in the range of preferably about 3 to about 8, more
preferably about 3.5 to about 7, and most preferably about 4 to
about 6, in particular about pH 5. The dry solids content is in the
range of preferably about 5 to about 50 wt %, more preferably about
10 to about 40 wt %, and most preferably about 20 to about 30 wt
%.
In the methods of the present invention, the enzyme composition can
comprise any protein that is useful in degrading or converting
cellulosic material.
In one aspect, the enzyme composition comprises one or more
(several) cellulolytic enzymes/proteins. In another aspect, the
enzyme composition comprises or further comprises one or more
(several) hemicellulolytic enzymes. In another aspect, the enzyme
composition comprises one or more (several) cellulolytic enzymes
and one or more (several) hemicellulolytic enzymes. In another
aspect, the enzyme composition comprises one or more (several)
enzymes selected from the group of cellulolytic enzymes and
hemicellulolytic enzymes.
In another aspect, the enzyme composition comprises one or more
(several) cellulolytic enzymes/proteins selected from the group
consisting of an endoglucanase, a cellobiohydrolase, a
beta-glucosidase, and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises or
further comprises one or more (several) proteins selected from the
group consisting of a hemicellulase, an expansin, an esterase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollenin. The hemicellulase is preferably one or more (several)
enzymes selected from the group consisting of an acetylmannan
esterase, an acetyxylan esterase, an arabinanase, an
arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase,
a galactosidase, a glucuronidase, a glucuronoyl esterase, a
mannanase, a mannosidase, a xylanase, and a xylosidase.
In another aspect, the enzyme composition comprises an
endoglucanase. In another aspect, the enzyme composition comprises
a cellobiohydrolase. In another aspect, the enzyme composition
comprises a beta-glucosidase. In another aspect, the enzyme
composition comprises a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises an
acetylmannan esterase. In another aspect, the enzyme composition
comprises an acetyxylan esterase. In another aspect, the enzyme
composition comprises an arabinanase (e.g., alpha-L-arabinanase).
In another aspect, the enzyme composition comprises an
arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). In another
aspect, the enzyme composition comprises a coumaric acid esterase.
In another aspect, the enzyme composition comprises a feruloyl
esterase. In another aspect, the enzyme composition comprises a
galactosidase (e.g., alpha-galactosidase and/or
beta-galactosidase). In another aspect, the enzyme composition
comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another
aspect, the enzyme composition comprises a glucuronoyl esterase. In
another aspect, the enzyme composition comprises a mannanase. In
another aspect, the enzyme composition comprises a mannosidase
(e.g., beta-mannosidase). In another aspect, the enzyme composition
comprises a xylanase. In a preferred aspect, the xylanase is a
Family 10 xylanase. In another aspect, the enzyme composition
comprises a xylosidase.
In another aspect, the enzyme composition comprises an expansin. In
another aspect, the enzyme composition comprises an esterase. In
another aspect, the enzyme composition comprises a ligninolytic
enzyme. In a preferred aspect, the ligninolytic enzyme is a
laccase. In another preferred aspect, the ligninolytic enzyme is a
manganese peroxidase. In another preferred aspect, the ligninolytic
enzyme is a lignin peroxidase. In another preferred aspect, the
ligninolytic enzyme is a H.sub.2O.sub.2-producing enzyme. In
another aspect, the enzyme composition comprises a pectinase. In
another aspect, the enzyme composition comprises a peroxidase. In
another aspect, the enzyme composition comprises a protease. In
another aspect, the enzyme composition comprises a swollenin.
In the methods of the present invention, the enzyme(s)/protein(s)
can be added prior to or during fermentation, e.g., during
saccharification or during or after propagation of the fermenting
microorganism(s).
One or more (several) components of the enzyme composition may be
wild-type proteins, recombinant proteins, or a combination of
wild-type proteins and recombinant proteins. For example, one or
more (several) components may be native proteins of a cell, which
is used as a host cell to express recombinantly one or more
(several) other components of the enzyme composition. One or more
(several) components of the enzyme composition may be produced as
monocomponents, which are then combined to form the enzyme
composition. The enzyme composition may be a combination of
multicomponent and monocomponent protein preparations.
The enzymes used in the methods of the present invention may be in
any form suitable for use in the processes described herein, such
as, for example, a crude fermentation broth with or without cells
removed, a cell lysate with or without cellular debris, a
semi-purified or purified enzyme preparation, or a host cell as a
source of the enzymes. The enzyme composition may be a dry powder
or granulate, a non-dusting granulate, a liquid, a stabilized
liquid, or a stabilized protected enzyme. Liquid enzyme
preparations may, for instance, be stabilized by adding stabilizers
such as a sugar, a sugar alcohol or another polyol, and/or lactic
acid or another organic acid according to established
processes.
The optimum amounts of the enzymes and polypeptides having
cellobiohydrolase activity depend on several factors including, but
not limited to, the mixture of component cellulolytic enzymes, the
cellulosic substrate, the concentration of cellulosic substrate,
the pretreatment(s) of the cellulosic substrate, temperature, time,
pH, and inclusion of fermenting organism (e.g., yeast for
Simultaneous Saccharification and Fermentation).
In a preferred aspect, an effective amount of cellulolytic protein
to cellulosic material is about 0.5 to about 50 mg, preferably
about 0.5 to about 40 mg, more preferably about 0.5 to about 25 mg,
more preferably about 0.75 to about 20 mg, more preferably about
0.75 to about 15 mg, even more preferably bout 0.5 to about 10 mg,
and most preferably about 2.5 to about 10 mg per g of cellulosic
material.
In another preferred aspect, an effective amount of polypeptide(s)
having cellobiohydrolase activity to cellulosic material is about
0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, more
preferably about 0.01 to about 30 mg, more preferably about 0.01 to
about 20 mg, more preferably about 0.01 to about 10 mg, more
preferably about 0.01 to about 5 mg, more preferably about 0.025 to
about 1.5 mg, more preferably about 0.05 to about 1.25 mg, more
preferably about 0.075 to about 1.25 mg, more preferably about 0.1
to about 1.25 mg, even more preferably about 0.15 to about 1.25 mg,
and most preferably about 0.25 to about 1.0 mg per g of cellulosic
material.
In another preferred aspect, an effective amount of polypeptide(s)
having cellobiohydrolase activity to cellulolytic protein is about
0.005 to about 1.0 g, preferably about 0.01 to about 1.0 g, more
preferably about 0.15 to about 0.75 g, more preferably about 0.15
to about 0.5 g, more preferably about 0.1 to about 0.5 g, even more
preferably about 0.1 to about 0.25 g, and most preferably about
0.05 to about 0.2 g per g of cellulolytic protein.
The enzymes can be derived or obtained from any suitable origin,
including, bacterial, fungal, yeast, plant, or mammalian origin.
The term "obtained" means herein that the enzyme may have been
isolated from an organism that naturally produces the enzyme as a
native enzyme. The term "obtained" also means herein that the
enzyme may have been produced recombinantly in a host organism
employing methods described herein, wherein the recombinantly
produced enzyme is either native or foreign to the host organism or
has a modified amino acid sequence, e.g., having one or more
(several) amino acids that are deleted, inserted and/or
substituted, i.e., a recombinantly produced enzyme that is a mutant
and/or a fragment of a native amino acid sequence or an enzyme
produced by nucleic acid shuffling processes known in the art.
Encompassed within the meaning of a native enzyme are natural
variants and within the meaning of a foreign enzyme are variants
obtained recombinantly, such as by site-directed mutagenesis or
shuffling.
The polypeptide having enzyme activity may be a bacterial
polypeptide. For example, the polypeptide may be a gram positive
bacterial polypeptide such as a Bacillus, Streptococcus,
Streptomyces, Staphylococcus, Enterococcus, Lactobacillus,
Lactococcus, Clostridium, Geobacillus, or Oceanobacillus
polypeptide having enzyme activity, or a Gram negative bacterial
polypeptide such as an E. coli, Pseudomonas, Salmonella,
Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,
Ilyobacter, Neisseria, or Ureaplasma polypeptide having enzyme
activity.
In a preferred aspect, the polypeptide is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide having enzyme
activity.
In another preferred aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme
activity.
In another preferred aspect, the polypeptide is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide having
enzyme activity.
The polypeptide having enzyme activity may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide having enzyme activity; or more preferably
a filamentous fungal polypeptide such as an Acremonium, Agaricus,
Alternaria, Aspergillus, Aureobasidium, Botryospaeria,
Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,
Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,
Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,
Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,
Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide
having enzyme activity.
In a preferred aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having enzyme activity.
In another preferred aspect, the polypeptide is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium
inops, Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium funiculosum,
Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, Trichoderma viride, or Trichophaea saccata
polypeptide having enzyme activity.
Chemically modified or protein engineered mutants of the
polypeptides having enzyme activity may also be used.
One or more (several) components of the enzyme composition may be a
recombinant component, i.e., produced by cloning of a DNA sequence
encoding the single component and subsequent cell transformed with
the DNA sequence and expressed in a host (see, for example, WO
91/17243 and WO 91/17244). The host is preferably a heterologous
host (enzyme is foreign to host), but the host may under certain
conditions also be a homologous host (enzyme is native to host).
Monocomponent cellulolytic proteins may also be prepared by
purifying such a protein from a fermentation broth.
Examples of commercial cellulolytic protein preparations suitable
for use in the present invention include, for example, CELLIC.TM.
Ctec (Novozymes A/S), CELLUCLAST.TM. (Novozymes A/S), NOVOZYM.TM.
188 (Novozymes A/S), CELLUZYME.TM. (Novozymes A/S), CEREFLO.TM.
(Novozymes A/S), and ULTRAFLO.TM. (Novozymes A/S), ACCELERASE.TM.
(Genencor Int.), LAMINEX.TM. (Genencor Int.), SPEZYME.TM. CP
(Genencor Int.), ROHAMENT.TM. 7069 W (Rohm GmbH), FIBREZYME.RTM.
LDI (Dyadic International, Inc.), FIBREZYME.RTM. LBR (Dyadic
International, Inc.), or VISCOSTAR.RTM. 150L (Dyadic International,
Inc.). The cellulase enzymes are added in amounts effective from
about 0.001 to about 5.0 wt % of solids, more preferably from about
0.025 to about 4.0 wt % of solids, and most preferably from about
0.005 to about 2.0 wt % of solids. The cellulase enzymes are added
in amounts effective from about 0.001 to about 5.0 wt % of solids,
more preferably from about 0.025 to about 4.0 wt % of solids, and
most preferably from about 0.005 to about 2.0 wt % of solids.
Examples of bacterial endoglucanases that can be used in the
methods of the present invention, include, but are not limited to,
an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO
93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No.
5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca
endoglucanase III (WO 05/093050); and Thermobifida fusca
endoglucanase V (WO 05/093050).
Examples of fungal endoglucanases that can be used in the methods
of the present invention, include, but are not limited to, a
Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45:
253-263; GENBANK.TM. accession no. M15665); Trichoderma reesei
endoglucanase II (Saloheimo, et al., 1988, Gene 63:11-22;
GENBANK.TM. accession no. M19373); Trichoderma reesei endoglucanase
III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563;
GENBANK.TM. accession no. AB003694); and Trichoderma reesei
endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13:
219-228; GENBANK.TM. accession no. Z33381); Aspergillus aculeatus
endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884);
Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current
Genetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti
et al., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase
(GENBANK.TM. accession no. L29381); Humicola grisea var. thermoidea
endoglucanase (GENBANK.TM. accession no. AB003107); Melanocarpus
albomyces endoglucanase (GENBANK.TM. accession no. MAL515703);
Neurospora crassa endoglucanase (GENBANK.TM. accession no.
XM.sub.--324477); Humicola insolens endoglucanase V; Myceliophthora
thermophila CBS 117.65 endoglucanase; basidiomycete CBS 495.95
endoglucanase; basidiomycete CBS 494.95 endoglucanase; Thielavia
terrestris NRRL 8126 CEL6B endoglucanase; Thielavia terrestris NRRL
8126 CEL6C endoglucanase); Thielavia terrestris NRRL 8126 CEL7C
endoglucanase; Thielavia terrestris NRRL 8126 CEL7E endoglucanase;
Thielavia terrestris NRRL 8126 CEL7F endoglucanase; Cladorrhinum
foecundissimum ATCC 62373 CEL7A endoglucanase; and Trichoderma
reesei strain No. VTT-D-80133 endoglucanase (GENBANK.TM. accession
no. M15665).
Examples of cellobiohydrolases useful in the methods of the present
invention include, but are not limited to, Trichoderma reesei
cellobiohydrolase I; Trichoderma reesei cellobiohydrolase II;
Humicola insolens cellobiohydrolase I, Myceliophthora thermophila
cellobiohydrolase II, Thielavia terrestris cellobiohydrolase II
(CEL6A), Chaetomium thermophilum cellobiohydrolase I, and
Chaetomium thermophilum cellobiohydrolase II.
Examples of beta-glucosidases useful in the methods of the present
invention include, but are not limited to, Aspergillus oryzae
beta-glucosidase; Aspergillus fumigatus beta-glucosidase;
Penicillium brasilianum IBT 20888 beta-glucosidase; Aspergillus
niger beta-glucosidase; and Aspergillus aculeatus
beta-glucosidase.
The Aspergillus oryzae polypeptide having beta-glucosidase activity
can be obtained according to WO 2002/095014. The Aspergillus
fumigatus polypeptide having beta-glucosidase activity can be
obtained according to WO 2005/047499. The Penicillium brasilianum
polypeptide having beta-glucosidase activity can be obtained
according to WO 2007/019442. The Aspergillus niger polypeptide
having beta-glucosidase activity can be obtained according to Dan
et al., 2000, J. Biol. Chem. 275: 4973-4980. The Aspergillus
aculeatus polypeptide having beta-glucosidase activity can be
obtained according to Kawaguchi et al., 1996, Gene 173:
287-288.
The beta-glucosidase may be a fusion protein. In one aspect, the
beta-glucosidase is the Aspergillus oryzae beta-glucosidase variant
BG fusion protein or the Aspergillus oryzae beta-glucosidase fusion
protein obtained according to WO 2008/057637.
Other endoglucanases, cellobiohydrolases, and beta-glucosidases are
disclosed in numerous Glycosyl Hydrolase families using the
classification according to Henrissat B., 1991, A classification of
glycosyl hydrolases based on amino-acid sequence similarities,
Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996,
Updating the sequence-based classification of glycosyl hydrolases,
Biochem. J. 316: 695-696.
Other cellulolytic enzymes that may be used in the present
invention are described in EP 495,257, EP 531,315, EP 531,372, WO
89/09259, WO 94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO
96/034108, WO 97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO
98/015619, WO 98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO
99/025846, WO 99/025847, WO 99/031255, WO 2000/009707, WO
2002/050245, WO 2002/0076792, WO 2002/101078, WO 2003/027306, WO
2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO
2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO
2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO
2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO
2008/008070, WO 2008/008793, U.S. Pat. Nos. 4,435,307, 5,457,046,
5,648,263, 5,686,593, 5,691,178, 5,763,254, and 5,776,757.
In the methods of the present invention, any GH61 polypeptide
having cellulolytic enhancing activity can be used.
In a first aspect, the polypeptide having cellulolytic enhancing
activity comprises the following motifs:
[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and
[FW]-[TF]-K-[AIV],
wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5
contiguous positions, and X(4) is any amino acid at 4 contiguous
positions.
The polypeptide comprising the above-noted motifs may further
comprise:
H-X(1,2)-G-P-X(3)-[YW]-[AILMV],
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or
H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],
wherein X is any amino acid, X(1,2) is any amino acid at 1 position
or 2 contiguous positions, X(3) is any amino acid at 3 contiguous
positions, and X(2) is any amino acid at 2 contiguous positions. In
the above motifs, the accepted IUPAC single letter amino acid
abbreviation is employed.
In a preferred aspect, the polypeptide having cellulolytic
enhancing activity further comprises
H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In another preferred aspect, the
isolated polypeptide having cellulolytic enhancing activity further
comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. In another
preferred aspect, the polypeptide having cellulolytic enhancing
activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].
In a second aspect, the polypeptide having cellulolytic enhancing
activity comprises the following motif:
[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ],
wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5
contiguous positions, and x(3) is any amino acid at 3 contiguous
positions. In the above motif, the accepted IUPAC single letter
amino acid abbreviation is employed.
Examples of polypeptides having cellulolytic enhancing activity
useful in the methods of the present invention include, but are not
limited to, polypeptides having cellulolytic enhancing activity
from Thielavia terrestris (WO 2005/074647); polypeptides having
cellulolytic enhancing activity from Thermoascus aurantiacus (WO
2005/074656); polypeptides having cellulolytic enhancing activity
from Trichoderma reesei (WO 2007/089290); and polypeptides having
cellulolytic enhancing activity from Myceliophthora thermophila (WO
2009/085935; WO 2009/085859; WO 2009/085864; WO 2009/085868).
Examples of commercial hemicellulolytic enzyme preparations (e.g.,
xylan degrading) suitable for use in the present invention include,
for example, SHEARZYME.TM. (Novozymes A/S), CELLIC.TM. Htec
(Novozymes A/S), VISCOZYME.RTM. (Novozymes A/S), ULTRAFLO.RTM.
(Novozymes A/S), PULPZYME.RTM. HC (Novozymes A/S), MULTIFECT.RTM.
Xylanase (Genencor), ECOPULP.RTM. TX-200A (AB Enzymes), HSP 6000
Xylanase (DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales, UK),
DEPOL.TM. 740L. (Biocatalysts Limit, Wales, UK), and DEPOL.TM. 762P
(Biocatalysts Limit, Wales, UK).
Examples of xylanases useful in the methods of the present
invention include, but are not limited to, Aspergillus aculeatus
xylanase (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus
xylanases (WO 2006/078256), and Thielavia terrestris NRRL 8126
xylanases (WO 2009/079210).
Examples of beta-xylosidases useful in the methods of the present
invention include, but are not limited to, Trichoderma reesei
beta-xylosidase (UniProtKB/TrEMBL accession number Q92458),
Talaromyces emersonii (SwissProt accession number Q8X212), and
Neurospora crassa (SwissProt accession number Q7SOW4).
Examples of acetylxylan esterases useful in the methods of the
present invention include, but are not limited to, Hypocrea
jecorina acetylxylan esterase (WO 2005/001036), Neurospora crassa
acetylxylan esterase (UniProt accession number q7s259), Thielavia
terrestris NRRL 8126 acetylxylan esterase (WO 2009/042846),
Chaetomium globosum acetylxylan esterase (Uniprot accession number
Q2GWX4), Chaetomium gracile acetylxylan esterase (GeneSeqP
accession number AAB82124), Phaeosphaeria nodorum acetylxylan
esterase (Uniprot accession number QOUHJ1), and Humicola insolens
DSM 1800 acetylxylan esterase (WO 2009/073709).
Examples of ferulic acid esterases useful in the methods of the
present invention include, but are not limited to, Humicola
insolens DSM 1800 feruloyl esterase (WO 2009/076122), Neurospora
crassa feruloyl esterase (UniProt accession number Q9HGR3), and
Neosartorya fischeri feruloyl esterase (UniProt Accession number
A1D9T4).
Examples of arabinofuranosidases useful in the methods of the
present invention include, but are not limited to, Humicola
insolens DSM 1800 arabinofuranosidase (WO 2009/073383) and
Aspergillus niger arabinofuranosidase (GeneSeqP accession number
AAR94170).
Examples of alpha-glucuronidases useful in the methods of the
present invention include, but are not limited to, Aspergillus
clavatus alpha-glucuronidase (UniProt accession number alcc12),
Trichoderma reesei alpha-glucuronidase (Uniprot accession number
Q99024), Talaromyces emersonii alpha-glucuronidase (UniProt
accession number Q8X211), Aspergillus niger alpha-glucuronidase
(Uniprot accession number Q96WX9), Aspergillus terreus
alpha-glucuronidase (SwissProt accession number Q0CJP9), and
Aspergillus fumigatus alpha-glucuronidase (SwissProt accession
number Q4WW45).
The enzymes and proteins used in the methods of the present
invention may be produced by fermentation of the above-noted
microbial strains on a nutrient medium containing suitable carbon
and nitrogen sources and inorganic salts, using procedures known in
the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More Gene
Manipulations in Fungi, Academic Press, CA, 1991). Suitable media
are available from commercial suppliers or may be prepared
according to published compositions (e.g., in catalogues of the
American Type Culture Collection). Temperature ranges and other
conditions suitable for growth and enzyme production are known in
the art (see, e.g., Bailey, J. E., and Ollis, D. F., Biochemical
Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).
The fermentation can be any method of cultivation of a cell
resulting in the expression or isolation of an enzyme. Fermentation
may, therefore, be understood as comprising shake flask
cultivation, or small- or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermentors performed in a suitable medium
and under conditions allowing the enzyme to be expressed or
isolated. The resulting enzymes produced by the methods described
above may be recovered from the fermentation medium and purified by
conventional procedures.
Fermentation. The fermentable sugars obtained from the hydrolyzed
cellulosic material can be fermented by one or more (several)
fermenting microorganisms capable of fermenting the sugars directly
or indirectly into a desired fermentation product. "Fermentation"
or "fermentation process" refers to any fermentation process or any
process comprising a fermentation step. Fermentation processes also
include fermentation processes used in the consumable alcohol
industry (e.g., beer and wine), dairy industry (e.g., fermented
dairy products), leather industry, and tobacco industry. The
fermentation conditions depend on the desired fermentation product
and fermenting organism and can easily be determined by one skilled
in the art.
In the fermentation step, sugars, released from cellulosic material
as a result of the pretreatment and enzymatic hydrolysis steps, are
fermented to a product, e.g., ethanol, by a fermenting organism,
such as yeast. Hydrolysis (saccharification) and fermentation can
be separate or simultaneous, as described herein.
Any suitable hydrolyzed cellulosic material can be used in the
fermentation step in practicing the present invention. The material
is generally selected based on the desired fermentation product,
i.e., the substance to be obtained from the fermentation, and the
process employed, as is well known in the art.
The term "fermentation medium" is understood herein to refer to a
medium before the fermenting microorganism(s) is(are) added, such
as, a medium resulting from a saccharification process, as well as
a medium used in a simultaneous saccharification and fermentation
process (SSF).
"Fermenting microorganism" refers to any microorganism, including
bacterial and fungal organisms, suitable for use in a desired
fermentation process to produce a fermentation product. The
fermenting organism can be C.sub.6 and/or C.sub.5 fermenting
organisms, or a combination thereof. Both C.sub.6 and C.sub.5
fermenting organisms are well known in the art. Suitable fermenting
microorganisms are able to ferment, i.e., convert, sugars, such as
glucose, xylose, xylulose, arabinose, maltose, mannose, galactose,
or oligosaccharides, directly or indirectly into the desired
fermentation product.
Examples of bacterial and fungal fermenting organisms producing
ethanol are described by Lin et al., 2006, Appl. Microbiol.
Biotechnol. 69: 627-642.
Examples of fermenting microorganisms that can ferment C.sub.6
sugars include bacterial and fungal organisms, such as yeast.
Preferred yeast includes strains of the Saccharomyces spp.,
preferably Saccharomyces cerevisiae.
Examples of fermenting organisms that can ferment C.sub.5 sugars
include bacterial and fungal organisms, such as yeast. Preferred
C.sub.5 fermenting yeast include strains of Pichia, preferably
Pichia stipitis, such as Pichia stipitis CBS 5773; strains of
Candida, preferably Candida boidinii, Candida brassicae, Candida
sheatae, Candida diddensii, Candida pseudotropicalis, or Candida
utilis.
Other fermenting organisms include strains of Zymomonas, such as
Zymomonas mobilis; Hansenula, such as Hansenula anomala;
Kluyveromyces, such as K. fragilis; Schizosaccharomyces, such as S.
pombe; and E. coli, especially E. coli strains that have been
genetically modified to improve the yield of ethanol.
In a preferred aspect, the yeast is a Saccharomyces spp. In a more
preferred aspect, the yeast is Saccharomyces cerevisiae. In another
more preferred aspect, the yeast is Saccharomyces distaticus. In
another more preferred aspect, the yeast is Saccharomyces uvarum.
In another preferred aspect, the yeast is a Kluyveromyces. In
another more preferred aspect, the yeast is Kluyveromyces
marxianus. In another more preferred aspect, the yeast is
Kluyveromyces fragilis. In another preferred aspect, the yeast is a
Candida. In another more preferred aspect, the yeast is Candida
boidinii. In another more preferred aspect, the yeast is Candida
brassicae. In another more preferred aspect, the yeast is Candida
diddensii. In another more preferred aspect, the yeast is Candida
pseudotropicalis. In another more preferred aspect, the yeast is
Candida utilis. In another preferred aspect, the yeast is a
Clavispora. In another more preferred aspect, the yeast is
Clavispora lusitaniae. In another more preferred aspect, the yeast
is Clavispora opuntiae. In another preferred aspect, the yeast is a
Pachysolen. In another more preferred aspect, the yeast is
Pachysolen tannophilus. In another preferred aspect, the yeast is a
Pichia. In another more preferred aspect, the yeast is a Pichia
stipitis. In another preferred aspect, the yeast is a
Bretannomyces. In another more preferred aspect, the yeast is
Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulose
bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
D.C., 179-212).
Bacteria that can efficiently ferment hexose and pentose to ethanol
include, for example, Zymomonas mobilis and Clostridium
thermocellum (Philippidis, 1996, supra).
In a preferred aspect, the bacterium is a Zymomonas. In a more
preferred aspect, the bacterium is Zymomonas mobilis. In another
preferred aspect, the bacterium is a Clostridium. In another more
preferred aspect, the bacterium is Clostridium thermocellum.
Commercially available yeast suitable for ethanol production
includes, e.g., ETHANOL RED.TM. yeast (available from
Fermentis/Lesaffre, USA), FALI.TM. (available from Fleischmann's
Yeast, USA), SUPERSTART.TM. and THERMOSACC.TM. fresh yeast
(available from Ethanol Technology, WI, USA), BIOFERM.TM. AFT and
XR (available from NABC--North American Bioproducts Corporation,
GA, USA), GERT STRAND.TM. (available from Gert Strand AB, Sweden),
and FERMIOL.TM. (available from DSM Specialties).
In a preferred aspect, the fermenting microorganism has been
genetically modified to provide the ability to ferment pentose
sugars, such as xylose utilizing, arabinose utilizing, and xylose
and arabinose co-utilizing microorganisms.
The cloning of heterologous genes into various fermenting
microorganisms has led to the construction of organisms capable of
converting hexoses and pentoses to ethanol (cofermentation) (Chen
and Ho, 1993, Cloning and improving the expression of Pichia
stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.
Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically
engineered Saccharomyces yeast capable of effectively cofermenting
glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter
and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae,
Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995,
Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing
the TKL1 and TAL1 genes encoding the pentose phosphate pathway
enzymes transketolase and transaldolase, Appl. Environ. Microbiol.
61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering
of Saccharomyces cerevisiae for efficient anaerobic xylose
fermentation: a proof of principle, FEMS Yeast Research 4: 655-664;
Beall et al., 1991, Parametric studies of ethanol production from
xylose and other sugars by recombinant Escherichia coli, Biotech.
Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of
bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214;
Zhang et al., 1995, Metabolic engineering of a pentose metabolism
pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243;
Deanda et al., 1996, Development of an arabinose-fermenting
Zymomonas mobilis strain by metabolic pathway engineering, Appl.
Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose
isomerase).
In a preferred aspect, the genetically modified fermenting
microorganism is Saccharomyces cerevisiae. In another preferred
aspect, the genetically modified fermenting microorganism is
Zymomonas mobilis. In another preferred aspect, the genetically
modified fermenting microorganism is Escherichia coli. In another
preferred aspect, the genetically modified fermenting microorganism
is Klebsiella oxytoca. In another preferred aspect, the genetically
modified fermenting microorganism is Kluyveromyces sp.
It is well known in the art that the organisms described above can
also be used to produce other substances, as described herein.
The fermenting microorganism is typically added to the degraded
lignocellulose or hydrolysate and the fermentation is performed for
about 8 to about 96 hours, such as about 24 to about 60 hours. The
temperature is typically between about 26.degree. C. to about
60.degree. C., in particular about 32.degree. C. or 50.degree. C.,
and at about pH 3 to about pH 8, such as around pH 4-5, 6, or
7.
In a preferred aspect, the yeast and/or another microorganism is
applied to the degraded cellulosic material and the fermentation is
performed for about 12 to about 96 hours, such as typically 24-60
hours. In a preferred aspect, the temperature is preferably between
about 20.degree. C. to about 60.degree. C., more preferably about
25.degree. C. to about 50.degree. C., and most preferably about
32.degree. C. to about 50.degree. C., in particular about
32.degree. C. or 50.degree. C., and the pH is generally from about
pH 3 to about pH 7, preferably around pH 4-7. However, some
fermenting organisms, e.g., bacteria, have higher fermentation
temperature optima. Yeast or another microorganism is preferably
applied in amounts of approximately 10.sup.5 to 10.sup.12,
preferably from approximately 10.sup.7 to 10.sup.10, especially
approximately 2.times.10.sup.8 viable cell count per ml of
fermentation broth. Further guidance in respect of using yeast for
fermentation can be found in, e.g., "The Alcohol Textbook" (Editors
K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University
Press, United Kingdom 1999), which is hereby incorporated by
reference.
For ethanol production, following the fermentation the fermented
slurry is distilled to extract the ethanol. The ethanol obtained
according to the methods of the invention can be used as, e.g.,
fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or
industrial ethanol.
A fermentation stimulator can be used in combination with any of
the processes described herein to further improve the fermentation
process, and in particular, the performance of the fermenting
microorganism, such as, rate enhancement and ethanol yield. A
"fermentation stimulator" refers to stimulators for growth of the
fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, for example, Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process, Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
Fermentation products: A fermentation product can be any substance
derived from the fermentation. The fermentation product can be,
without limitation, an alcohol (e.g., arabinitol, butanol, ethanol,
glycerol, methanol, 1,3-propanediol, sorbitol, and xylitol); an
organic acid (e.g., acetic acid, acetonic acid, adipic acid,
ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic
acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,
glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid,
malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic
acid, succinic acid, and xylonic acid); a ketone (e.g., acetone);
an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,
serine, and threonine); and a gas (e.g., methane, hydrogen
(H.sub.2), carbon dioxide (CO.sub.2), and carbon monoxide (CO)).
The fermentation product can also be protein as a high value
product.
In a preferred aspect, the fermentation product is an alcohol. It
will be understood that the term "alcohol" encompasses a substance
that contains one or more hydroxyl moieties. In a more preferred
aspect, the alcohol is arabinitol. In another more preferred
aspect, the alcohol is butanol. In another more preferred aspect,
the alcohol is ethanol. In another more preferred aspect, the
alcohol is glycerol. In another more preferred aspect, the alcohol
is methanol. In another more preferred aspect, the alcohol is
1,3-propanediol. In another more preferred aspect, the alcohol is
sorbitol. In another more preferred aspect, the alcohol is xylitol.
See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,
1999, Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira,
M. M., and Jonas, R., 2002, The biotechnological production of
sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and
Singh, D., 1995, Processes for fermentative production of
xylitol--a sugar substitute, Process Biochemistry 30 (2): 117-124;
Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of
acetone, butanol and ethanol by Clostridium beijerinckii BA101 and
in situ recovery by gas stripping, World Journal of Microbiology
and Biotechnology 19 (6): 595-603.
In another preferred aspect, the fermentation product is an organic
acid. In another more preferred aspect, the organic acid is acetic
acid. In another more preferred aspect, the organic acid is
acetonic acid. In another more preferred aspect, the organic acid
is adipic acid. In another more preferred aspect, the organic acid
is ascorbic acid. In another more preferred aspect, the organic
acid is citric acid. In another more preferred aspect, the organic
acid is 2,5-diketo-D-gluconic acid. In another more preferred
aspect, the organic acid is formic acid. In another more preferred
aspect, the organic acid is fumaric acid. In another more preferred
aspect, the organic acid is glucaric acid. In another more
preferred aspect, the organic acid is gluconic acid. In another
more preferred aspect, the organic acid is glucuronic acid. In
another more preferred aspect, the organic acid is glutaric acid.
In another preferred aspect, the organic acid is 3-hydroxypropionic
acid. In another more preferred aspect, the organic acid is
itaconic acid. In another more preferred aspect, the organic acid
is lactic acid. In another more preferred aspect, the organic acid
is malic acid. In another more preferred aspect, the organic acid
is malonic acid. In another more preferred aspect, the organic acid
is oxalic acid. In another more preferred aspect, the organic acid
is propionic acid. In another more preferred aspect, the organic
acid is succinic acid. In another more preferred aspect, the
organic acid is xylonic acid. See, for example, Chen, R., and Lee,
Y. Y., 1997, Membrane-mediated extractive fermentation for lactic
acid production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
In another preferred aspect, the fermentation product is a ketone.
It will be understood that the term "ketone" encompasses a
substance that contains one or more ketone moieties. In another
more preferred aspect, the ketone is acetone. See, for example,
Qureshi and Blaschek, 2003, supra.
In another preferred aspect, the fermentation product is an amino
acid. In another more preferred aspect, the organic acid is
aspartic acid. In another more preferred aspect, the amino acid is
glutamic acid. In another more preferred aspect, the amino acid is
glycine. In another more preferred aspect, the amino acid is
lysine. In another more preferred aspect, the amino acid is serine.
In another more preferred aspect, the amino acid is threonine. See,
for example, Richard, A., and Margaritis, A., 2004, Empirical
modeling of batch fermentation kinetics for poly(glutamic acid)
production and other microbial biopolymers, Biotechnology and
Bioengineering 87 (4): 501-515.
In another preferred aspect, the fermentation product is a gas. In
another more preferred aspect, the gas is methane. In another more
preferred aspect, the gas is H.sub.2. In another more preferred
aspect, the gas is CO.sub.2. In another more preferred aspect, the
gas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama,
1997, Studies on hydrogen production by continuous culture system
of hydrogen-producing anaerobic bacteria, Water Science and
Technology 36 (6-7): 41-47; and Gunaseelan V. N. in Biomass and
Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic digestion of
biomass for methane production: A review.
Recovery. The fermentation product(s) can be optionally recovered
from the fermentation medium using any method known in the art
including, but not limited to, chromatography, electrophoretic
procedures, differential solubility, distillation, or extraction.
For example, alcohol is separated from the fermented cellulosic
material and purified by conventional methods of distillation.
Ethanol with a purity of up to about 96 vol. % can be obtained,
which can be used as, for example, fuel ethanol, drinking ethanol,
i.e., potable neutral spirits, or industrial ethanol.
Signal Peptide
The present invention also relates to an isolated polynucleotide
encoding a signal peptide comprising or consisting of amino acids 1
to 17 of SEQ ID NO: 2 or amino acids 1 to 17 of SEQ ID NO: 4. The
polynucleotide may further comprise a gene encoding a protein,
which is operably linked to the signal peptide. The protein is
preferably foreign to the signal peptide.
The present invention also relates to nucleic acid constructs,
expression vectors and recombinant host cells comprising such
polynucleotides.
The present invention also relates to methods of producing a
protein, comprising: (a) cultivating a recombinant host cell
comprising such polynucleotide; and (b) recovering the protein.
The protein may be native or heterologous to a host cell. The term
"protein" is not meant herein to refer to a specific length of the
encoded product and, therefore, encompasses peptides,
oligopeptides, and polypeptides. The term "protein" also
encompasses two or more polypeptides combined to form the encoded
product. The proteins also include hybrid polypeptides and fused
polypeptides.
Preferably, the protein is a hormone or variant thereof, enzyme,
receptor or portion thereof, antibody or portion thereof, or
reporter. For example, the protein may be an oxidoreductase,
transferase, hydrolase, lyase, isomerase, or ligase such as an
aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, esterase, alpha-galactosidase,
beta-galactosidase, glucoamylase, alpha-glucosidase,
beta-glucosidase, invertase, laccase, another lipase, mannosidase,
mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase or xylanase.
The gene may be obtained from any prokaryotic, eukaryotic, or other
source.
The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
Chemicals used as buffers and substrates were commercial products
of at least reagent grade.
Media
YG agar plates were composed of 5.0 g of Difco powdered yeast
extract, 10.0 g of glucose, 20.0 g of agar, and deionized water to
1 liter.
PDA plates were composed of 39 g of potato dextrose agar and
deionized water to 1 liter.
Minimal medium plates were composed of 6 g of NaNO.sub.3, 0.52 g of
KCl, 1.52 g of KH.sub.2PO.sub.4, 1 ml of COVE trace elements
solution, 20 g of Noble agar, 20 ml of 50% glucose, 2.5 ml of
MgSO.sub.4.7H.sub.2O, 20 ml of a 0.02% biotin solution, and
deionized water to 1 liter.
COVE trace elements solution was composed of 0.04 g of
Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O,
1.2 g of FeSO.sub.4.7H.sub.2O, 0.7 g of MnSO.sub.4.H.sub.2O, 0.8 g
of Na.sub.2MoO.sub.2.2H.sub.2O, 10 g of ZnSO.sub.4.7H.sub.2O, and
deionized water to 1 liter.
YPM medium was composed of 2% yeast extract, 2% Peptone, and 1% of
maltose.
Example 1
Isolation of Thielavia hyrcania Strains NN045178 and NN045097
Thielavia hyrcania strains NN045178 and NN045097 were isolated from
soil samples collected in Yunnan China on Mar. 9, 1999. The strains
were isolated by applying a soil suspension solution on YG agar
plates followed by incubation at 45.degree. C. for 3-5 days and
further purification of fungal colonies. The strains were
identified by both morphological characters and DNA sequencing of
internal transcribed spacer (ITS) regions. Thielavia hyrcania
NN045178 was deposited with the China General Microbiological
Culture Collectio Center and assigned deposit number CGMCC
0864.
Example 2
Thielavia hyrcania NN045178 and NN045097 Genomic DNA Extraction
Both Thielavia hyrcania strains NN045178 and NN045097 were grown
separately on PDA agar plates at 45.degree. C. for 3 days. Mycelia
were each collected directly from the agar plates into sterilized
motars and frozen under liquid nitrogen. Frozen mycelia were
ground, by mortar and pestle, to a fine powder, and genomic DNA was
isolated using a DNEASY.RTM. Plant Mini Kit (QIAGEN Inc., Valencia,
Calif., USA).
Example 3
Isolation of Full-Length Family 6 Cellobiohydrolase Genes from
Thielavia hyrcania Strains NN045178 and NN045097
A partial sequence of a Family 6 cellobiohydrolase gene was
identified from Thielavia hyrcania NN045178 by PCR screening using
the probes shown below, which were derived from the conserved
regions of known Family 6 cellobiohydrolase genes.
TABLE-US-00001 Primer CBHIIDF1: (SEQ ID NO: 5)
t(gt)cc(tc)ga(tc)cg(tc)ga(tc)tg(tc)gc Primer CBHIIDR2: (SEQ ID NO:
6) tc(ag)ccacc(gt)ggctt(gt)a(tc)cca
A full-length Family 6 cellobiohydrolase gene was isolated from
Thielavia hyrcania NN045178 using a Genome Walking Kit (Takara Bio
Inc., Seta 3-4-1, Otsu, Shiga, 520-2193, Japan) according to the
manufacturer's instructions. Briefly, total genomic DNA from
Thielavia hyrcania NN045178 was used as template for PCR
amplifications of the 5' end and the 3' end of the gene. Two
gene-specific primers shown below were used for each cloning, one
for primary PCR and one for secondary PCR. The primers were
designed based on the partial Family 6 cellobiohydrolase gene
sequence from Thielavia hyrcania NN045178.
TABLE-US-00002 Primer CBHII45178DPf1:
5'-AACTACGACGAGAAGCACTACGTCGAG-3' (SEQ ID NO: 7) Primer
CBHII45178DPf3: 5'-AGCTCCTGGATGCCTTTGTCTGGA-3' (SEQ ID NO: 8)
Primer CBHII45178DPas1: 5'-AGGCGTTGTAGTTGGCGACGTTG-3' (SEQ ID NO:
9) Primer CBHII45178DPas3: 5'-CAATCGAGAACTCGCCGTTGGA-3' (SEQ ID NO:
10)
The primary amplifications were composed of 2 .mu.l of genomic DNA
as template, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, 100 pmol of
AP2 (Takara Bio Inc., Seta 3-4-1, Otsu, Shiga, 520-2193, Japan),
and 10 pmol of primer CBHII45178DPas1 for 5' end cloning or 10 pmol
of primer CBHII45178DPf1 for 3' end cloning, 5 .mu.l of 10.times.
LA PCR Buffer II (Takara Bio Inc., Seta 3-4-1, Otsu, Shiga,
520-2193, Japan), and 2.5 units of TakaRa LA Taq DNA polymerase
(Takara Bio Inc., Seta 3-4-1, Otsu, Shiga, 520-2193, Japan) in a
final volume of 50 .mu.l. The amplifications were performed using
an Peltier Thermal Cycler (Bio-Rad Laboratories, Inc. Hercules,
Calif., USA) programmed for pre-denaturing at 94.degree. C. for 1
minute and 98.degree. C. for 1 minute; five cycles each at a
denaturing temperature of 94.degree. C. for 30 seconds; annealing
at 60.degree. C. for 1 minute, and elongation at 72.degree. C. for
2 minutes; 1 cycle of denaturing at 94.degree. C. for 30 seconds;
annealing at 25.degree. C. for 3 minutes and elongation at
72.degree. C. for 2 minutes; fifteen repeats of 2 cycles at
94.degree. C. for 30 seconds; 62.degree. C. for 1 minutes; and
72.degree. C. for 2 minutes; followed by 1 cycle at 94.degree. C.
for 30 seconds; 44.degree. C. for 1 minutes; and 72.degree. C. for
2 minutes; and a final extension at 72.degree. C. for 10 minutes.
The heat block then went to a 4.degree. C. soak cycle.
The secondary ampliifications were composed of 2 .mu.l of 20.times.
diluted primary PCR product as template, 2.5 mM each of dATP, dTTP,
dGTP, and dCTP, 100 pmol of AP2, and 10 pmol of primer
CBHII45178DPas3 for 5' end cloning or 10 pmol of primer
CBHII45178DPf3 for 3' end cloning, 5 .mu.l of 10.times. LA PCR
Buffer II, and 2.5 units of TakaRa LA Taq DNA polymerase in a final
volume of 50 .mu.l. The amplifications were performed using an
Peltier Thermal Cycler programmed for fifteen repeats of 2 cycles
of 94.degree. C. for 30 seconds; 62.degree. C. for 1 minutes;
72.degree. C. for 2 minutes; followed by 1 cycle of 94.degree. C.
for 30 seconds; 44.degree. C. for 1 minutes; 72.degree. C. for 2
minutes; and a final extension at 72.degree. C. for 10 minutes. The
heat block then went to a 4.degree. C. soak cycle.
The reaction products were isolated by 1.0% agarose gel
electrophoresis using 90 mM Tris-borate and 1 mM EDTA (TBE) buffer
where a 600 by product band from the 3' end cloning reaction and a
1.1 kb product band from the 5' end cloning reaction were excised
from the gels, purified using an illustra GFX PCR DNA and Gel Band
Purification Kit (GE Healthcare, Buckinghamshire, UK) according to
the manufacturer's instructions, and confirmed by DNA sequencing
using a 3730XL DNA Analyzer (Applied Biosystems Inc, Foster City,
Calif., USA).
As the partial sequence of another Family 6 cellobiohydrolase gene
of Thielavia hyrcania NN045097 showed high identity (97%) to the
Thielavia hyrcania NN045178 cellobiohydrolase gene, the full length
gene of the Family 6 cellobiohydrolase gene of Thielavia hyrcania
NN045097 was cloned using the same primer pair as the cloning of
Thielavia hyrcania NN045178 cellobiohydrolase gene.
Example 4
Characterization of the Thielavia hyrcania NN045097 and NN045178
Genomic Sequences Encoding Family 6 Cellobiohydrolases
The nucleotide sequence of the full-length Thielavia hyrcaniea
NN045097 Family 6 cellobiohydrolase gene is 1598 bp including the
stop codon. The deduced protein sequence of the mature protein
showed similarity to the exoglucanase GH6A from Humicola insolens
(uniprot:Q9C1S9).
The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid
sequence (SEQ ID NO: 2) of the full length Thielavia hyrcaniea
NN045097 Family 6 cellobiohydrolase gene are shown in FIG. 1. The
genomic fragment encodes a polypeptide of 493 amino acids,
interrupted by 2 introns of 58 bp each. The % G+C content of the
gene and the mature coding sequence are each 65.7%. Using the
SignalP software program (Nielsen et al., 1997, supra), a signal
peptide of 17 residues was predicted. The predicted mature protein
contains 476 amino acids with a predicted molecular mass of 50.0
kDa.
A comparative pairwise global alignment of amino acid sequences was
determined using the Needleman-Wunsch algorithm (Needleman and
Wunsch, 1970, supra) as implemented in the Needle program of EMBOSS
with gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 matrix. The alignment showed that the deduced amino acid
sequence of the Thielavia hyrcaniea NN045097 Family 6
cellobiohydrolase gene encoding the mature polypeptide shares 84.1%
and 83.7% identity (excluding gaps) with the deduced amino acid
sequences of two lignocellulosic enzyme proteins (WO 2008/095033;
GeneSeqP accession numbers ASR94104 and ASR94110,
respectively).
The nucleotide sequence of the full-length Thielavia hyrcania
NN045178 Family 6 cellobiohydrolase gene is 1584 bp including the
stop codon. The deduced protein sequence of the mature protein
showed similarity to the exoglucanase GH6A from Humicola insolens
(uniprot:Q9C1S9).
The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid
sequence (SEQ ID NO: 4) of the full-length Thielavia hyrcania
NN045178 Family 6 cellobiohydrolase gene are shown in FIG. 2. The
genomic fragment encodes a polypeptide of 487 amino acids,
interrupted by 2 introns of 59 and 61 bp. The % G+C content of the
gene and the mature coding sequence are each 65.4%. Using the
SignalP software program (Nielsen et al., 1997, supra), a signal
peptide of 17 residues was predicted. The predicted mature protein
contains 470 amino acids with a predicted molecular mass of 49.5
kDa.
A comparative pairwise global alignment of amino acid sequences was
determined using the Needleman-Wunsch algorithm (Needleman and
Wunsch, 1970, supra) as implemented in the Needle program of EMBOSS
with gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 matrix. The alignment showed that the deduced amino acid
sequence of the Thielavia hyrcania NN045178 Family 6
cellobiohydrolase gene encoding the mature polypeptide shares 85.3%
and 83.6% identity (excluding gaps) with the deduced amino acid
sequences of two lignocellulosic enzyme proteins (WO 2008/095033;
GeneSeqP accession numbers ASR94104 and ASR94110,
respectively).
Example 5
Cloning of the Thielavia hyrcania NN045178 Family 6
Cellobiohydrolase Gene and Construction of an Aspergillus oryzae
Expression Vector
Two synthetic oligonucleotide primers shown below were designed to
PCR amplify the Thielavia hyrcania NN045178 cellobiohydrolase gene
from the genomic DNA prepared in Example 2. An IN-FUSION.RTM. CF
Dry-down Cloning Kit (Clontech Laboratories, Inc., Mountain View,
Calif., USA) was used to clone the fragment directly into the
expression vector pPFJO355, without the need for restriction
digestion and ligation. The expression vector pPFJO355 contains the
Aspergillus oryzae TAKA alpha-amylase promoter and the Aspergillus
niger glucoamylase terminator. Furthermore pPFJO355 has pUC18
derived sequences for selection and propagation in E. coli, and a
pyrG gene, which encodes an orotidine decarboxylase derived from
Aspergillus nidulans for selection of a transformant of a pyrG
mutant Aspergillus strain.
TABLE-US-00003 H_45178_IFNbam: (SEQ ID NO: 11)
5'-ACACAACTGGGGATCCACCATGGCCAAGAAGCTCCTTCTCACC-3' H_45178_IFCbgl:
(SEQ ID NO: 12) 5'-GTCACCCTCTAGATCTCTAAAAAGGAGGGTTGGCGTTGG-3'
Bold letters represent coding sequence. The remaining sequence is
homologous to the insertion sites of pPFJO355.
Ten picomoles of each of the primers above were used in a PCR
composed of Thielavia hyrcania NN045178 genomic DNA, 10 .mu.l of
5.times. GC Buffer (Finnzymes Oy, Espoo, Finland), 1.5 .mu.l of
DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 1 unit of
PHUSION.TM. High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo,
Finland) in a final volume of 50 .mu.l. The amplification was
performed using an Peltier Thermal Cycler programmed for 1 cycle at
98.degree. C. for 1 minute; 5 cycles of denaturing at 98.degree. C.
for 15 seconds, annealing at 63.degree. C. for 30 seconds, with a
1.degree. C. increase per cycle and elongation at 72.degree. C. for
80 seconds; and another 25 cycles each at 98.degree. C. for 15
seconds and 72.degree. C. for 80 seconds; final extension at
72.degree. C. for 10 minutes. The heat block then went to a
4.degree. C. soak cycle.
The reaction products were isolated by 1.0% agarose gel
electrophoresis using TBE buffer where an approximately 1.8 kb
product band was excised from the gel, and purified using an
illustra GFX PCR DNA and Gel Band Purification Kit according to the
manufacturer's instructions.
Plasmid pPFJO355 was digested with Bam I and Bgl II, isolated by
1.0% agarose gel electrophoresis using TBE buffer, and purified
using an illustra GFX PCR DNA and Gel Band Purification Kit
according to the manufacturer's instructions.
The gene fragment and the digested vector were ligated together
using an IN-FUSION.RTM. CF Dry-down PCR Cloning Kit resulting in
pCBHII45178 (FIG. 3) in which transcription of the
cellobiohydrolase gene was under the control of the Aspergillus
oryzae TAKA alpha-amylase promoter. In brief, 30 ng of pPFJO355
digested with Bam HI and Bgl II, and 80 ng of the Thielavia
hyrcania NN045178 Family 6 cellobiohydrolase gene purified PCR
product were added to a reaction vial and resuspended in a final
volume of 10 .mu.l with addition of deionized water. The reaction
was incubated at 37.degree. C. for 15 minutes and then 50.degree.
C. for 15 minutes. Three .mu.l of the reaction was used to
transform E. coli TOP10 competent cells (TIANGEN Biotech (Beijing)
Co. Ltd., Beijing, China). An E. coli transformant containing
pCBHII45178 was detected by colony PCR and plasmid DNA was prepared
using a QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, Calif.,
USA). The Thielavia hyrcania NN045178 Family 6 cellobiohydrolase
gene insert in pCBHII45178 was confirmed by DNA sequencing using a
3730XL DNA Analyzer (Applied Biosystems Inc, Foster City, Calif.,
USA).
The same PCR fragment was cloned into vector pGEM-T (Promega
Corporation, Madison, Wis., USA) using a pGEM-T Vector System
(Promega Corporation, Madison, Wis., USA) to generate
pGEM-T-CBHII45178 (FIG. 4). The Thielavia hyrcania NN045178 Family
6 cellobiohydrolase gene insert in T-CBHII45178 was confirmed by
DNA sequencing as above. E. coli T-CBHII45178 was deposited with
the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH
(DSM) on May 20, 2009 and assigned accession number DSM 22599.
Example 6
Cloning a Family 6 Cellobiohydrolase Gene from Thielavia hyrcania
NN045097 and Construction of an Aspergillus oryzae Expression
Vector
The cloning of the Family 6 cellobiohydrolase gene from Thielavia
hyrcania NN045097 was performed according to the same method
described in Example 5.
The two synthetic oligonucleotide primers (H.sub.--45178_IFNbam and
H.sub.--45178_IFCbgl) described in Example 5 were used for PCR
amplification of the full length Family 6 cellobiohydrolase gene
from Thielavia hyrcania NN045097.
Ten picomoles of each of the primers above were used in a PCR
composed of Thielavia hyrcaniea NN045097 genomic DNA, 10 .mu.l of
5.times. GC Buffer, 1.5 .mu.l of DMSO, 2.5 mM each of dATP, dTTP,
dGTP, and dCTP, and 1 unit of PHUSION.TM. High-Fidelity DNA
Polymerase in a final volume of 50 .mu.l. The amplification was
performed using an Peltier Thermal Cycler programmed for 1 cycle at
98.degree. C. for 1 minutes; 10 cycles of denaturing at 98.degree.
C. for 15 seconds, annealing at 62.degree. C. for 30 seconds, with
a 1.degree. C. increase per cycle and elongation at 72.degree. C.
for 80 seconds; and another 25 cycles each at 98.degree. C. for 15
seconds and 72.degree. C. for 80 seconds; final extension at
72.degree. C. for 10 minutes. The heat block then went to a
4.degree. C. soak cycle.
The reaction products were isolated by 1.0% agarose gel
electrophoresis using TBE buffer where an approximately 1.8 kb
product band was excised from the gel, and purified using an
illustra GFX PCR DNA and Gel Band Purification Kit according to the
manufacturer's instructions.
Plasmid pPFJO355 was digested with Bam I and Bgl II, isolated by
1.0% agarose gel electrophoresis using TBE buffer, and purified
using an illustra GFX PCR DNA and Gel Band Purification Kit
according to the manufacturer's instructions.
The gene fragment and the digested vector were ligated together
using an IN-FUSION.RTM. CF Dry-down PCR Cloning Kit resulting in
pCBHII45097 (FIG. 5) in which transcription of the
cellobiohydrolase gene was under the control of the Aspergillus
oryzae TAKA alpha-amylase promoter. In brief, 30 ng of pPFJO355
digested with Bam I and Bgl II and 80 ng of the Thielavia hyrcaniea
NN045097 Family 6 cellobiohydrolase gene purified PCR product were
added to a reaction vial and resuspended in a final volume of 10
.mu.l with addition of deionized water. The reaction was incubated
at 37.degree. C. for 15 minutes and then 50.degree. C. for 15
minutes. Three .mu.l of the reaction was used to transform E. coli
TOP10 competent cells. An E. coli transformant containing
pCBHII45097 was detected by colony PCR and plasmid DNA was prepared
using a QIAprep Spin Miniprep Kit. The Thielavia hyrcaniea NN045097
Family 6 cellobiohydrolase gene insert in pCBHII45178 was confirmed
by DNA sequencing using a 3730XL DNA Analyzer.
The same PCR fragment was cloned into vector pGEM-T using a pGEM-T
Vector System to generate pGEM-T-CBHII45097 (FIG. 6). The Thielavia
hyrcaniea NN045097 Family 6 cellobiohydrolase gene insert in
pGEM-T-CBHII45097 was confirmed by DNA sequencing. E. coli
T-CBHII45097 was deposited with Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSM) on May 20, 2009 and
assigned accession number DSM 22598.
Example 7
Expression of the Thielavia hyrcania NN045178 and NN45097 Family 6
Cellobiohydrolase Genes in Aspergillus oryzae HowB101
Aspergillus oryzae HowB101 (WO 95/35385) protoplasts were prepared
according to the method of Christensen et al., 1988, Bio/Technology
6: 1419-1422. Three .mu.g of pCBHII45178 or pCBHII45097 were used
to transform the Aspergillus oryzae HowB101 protoplasts.
The transformation of Aspergillus oryzae HowB101 with pCBHII45178
or pCBHII45097 yielded about 50 transformants each. Four
transformants from each transformation were isolated to individual
Minimal medium plates.
The four transformants from each transformation were inoculated
separately into 3 ml of YPM medium in 24-well plate and incubated
at 30.degree. C. with shaking at 150 rpm. After 3 days incubation,
20 .mu.l of supernatant from each culture were analyzed on a
NUPAGE.RTM. NOVEX.RTM. 4-12% Bis-Tris Gel with MES (Invitrogen
Corporation, Carlsbad, Calif., USA) according to the manufacturer's
instructions. The resulting gel was stained with INSTANTBLUE.TM.
(Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of the
cultures showed that the majority of the transformants produced a
major protein band at approximately 70 kDa for both genes. The
expression strains harboring the Thielavia hyrcania NN045178 Family
6 cellobiohydrolase gene and the Thielavia hyrcania NN045097 Family
6 cellobiohydrolase gene were designated EXP02989 and EXP03004,
respectively.
A slant for each expression strain, designated transformant 4 of
EXP02989 and transformant 16 of EXP03004, was washed with 10 ml of
YPM medium and each strain was inoculated into a 2 liter flask
containing 400 ml of YPM medium to generate broth for
characterization of each enzyme. The cultures were harvested on day
3 and filtered using a 0.45 .mu.m DURAPORE.RTM. Membrane
(Millipore, Bedford, Mass., USA).
Example 8
Characterization of the Thielavia hyrcaniae NN045178 CEL6A
Cellobiohydrolase
Activity assay on PASC. A PASO stock slurry solution was prepared
by moistening 5 g of AVICEL.RTM. (JRS Pharma LP, Patterson, N.Y.,
USA) with water, followed by the addition of 150 ml of ice cold 85%
o-phosphoric acid. The suspension was slowly stirred in an ice-bath
for 1 hour. Then 500 ml of ice cold acetone were added while
stirring. The slurry was filtered using MIRACLOTH.RTM. (Calbiochem,
La Jolla, Calif., USA) and then washed three times with 100 ml of
ice cold acetone (drained as dry as possible after each wash).
Finally, the filtered slurry was washed twice with 500 ml of water,
and again drained as dry as possible after each wash. The PASO was
mixed with deionized water to a total volume of 500 ml at a
concentration of 10 g/liter, blended to homogeneity (using an
ULTRA-TURRAX.RTM. Homogenizer, Cole-Parmer, Vernon Hills, Ill.,
USA), and stored in a refrigerator for up to one month.
The PASC stock solution was diluted with 50 mM sodium acetate pH
5.0 buffer to a concentration of 2 g/liter, and used as the
substrate. To 150 .mu.l of PASO stock solution, 20 .mu.l of enzyme
sample were added and the reaction mixture was incubated for 60
minutes with shaking at 850 rpm. At the end of the incubation, 50
.mu.l of 2% NaOH were added to stop the reaction. The reaction
mixture was centrifuged at 1,000.times.g. The released sugars were
measured by first mixing 10 .mu.l of the reaction mixture with 90
.mu.l of 0.4% NaOH, followed by 50 .mu.l of 1.5% p-hydroxybenzoic
acid hydrazide in 2% NaOH (PHBAH, Sigma Chemical Co., St. Louis,
Mo., USA). The mixture was boiled at 100.degree. C. for 5 minutes,
and then 100 .mu.l were transferred to a microtiter plate for an
absorbance reading at 410 nm (Spectra Max M2, Molecular devices
Sunnyvale, Calif., USA). Blanks were made by omitting PASC in the
hydrolysis step, and by replacing the hydrolysate with buffer in
the sugar determination step.
pH profile. In a microtiter plate, 20 .mu.l of each
cellobiohydrolase (EXP02989 or EXP03004) were mixed with 120 .mu.l
of buffer (100 mM succinic acid, HEPES, CHES, or CAPSO, and 1 mM
CaCl.sub.2, 150 mM KCl, and 0.01% TRITON.RTM. X-100; pH adjusted
with NaOH and HCl) at pH 3, 4, 5, 6, 7, or 8, followed by addition
of 30 .mu.l of 10 g/liter PASC. The mixtures were placed on ice
before reaction. The assays were initiated by transferring the
microtiter plates to an EPPENDORF.RTM. thermomixer (Eppendorf,
Hauppauge, N.Y., USA), which was set at 50.degree. C., and
incubated for 60 minutes with shaking at 850 rpm. At the end the
incubation, 50 .mu.l of 2% NaOH were added to stop the reactions.
The reaction mixtures were centrifuged at 1,000.times.g. The
released sugars were measured as described above.
The results shown in FIG. 7 indicated that the cellobiohydrolases
possess activity over a broad pH-range from pH 3 to 11. The optimum
pH for each cellobiohydrolase was around pH 5 to 6.
pH Stability. A 40 .mu.l sample of each cellobiohydrolase (EXP02989
or EXP03004) was mixed with 160 .mu.l of 100 mM buffer (pH 4, 5, 6,
7, or 8: 100 mM succinic acid, HEPES, CHES, CAPSO, and 1 mM
CaCl.sub.2, 150 mM KCl, and 0.01% TRITON.RTM. X-100, pH adjusted
with NaOH and HCl) and incubated at 50.degree. C. with shaking at
850 rpm. Samples of 30 .mu.l were removed and placed on ice at 0,
10, 30, 60 and 110 minutes. Then 150 .mu.l of 2 g/liter PASC (in 50
mM sodium acetate pH 5.0) were added to each sample, and incubated
at 50.degree. C., 850 rpm for 60 minutes. Thirty .mu.l of each
buffer were run as blank. The released sugars were measured as
described above.
The results shown in FIGS. 8A and 8B indicated that the EXP02989
cellobiohydrolase had residual activity at pH 4, 5, 6, 7, and 8 of
90%, 96%, 96%, 90%, and 39%, respectively, while the EXP03004
cellobiohydrolase had residual activity at pH 4, 5, 6, 7, and 8 of
89%, 93%, 95%, 75%, and 48%, respectively, after incubation for 110
minutes at 50.degree. C.
Temperature Profile. A 150 .mu.l volume of 2 g of PASC per liter of
50 mM sodium acetate pH 5.0 buffer in an EPPENDORF.RTM. tube was
preincubated at 25, 30, 40, 50, 60, 70, 80, 90, and 100.degree. C.
The assay was initiated by adding 20 .mu.l of each
cellobiohydrolase (EXP02989 or EXP03004) followed by incubation for
60 minutes with shaking at 850 rpm. At the end of the incubation,
50 .mu.l of 2% sodium hydroxide were added to stop the reaction and
the reaction mixtures were centrifuged at 1,000.times.g. The
released sugars were measured as described above.
The results shown in FIGS. 9A and 9B indicated that the
cellobiohydrolases were active over a wide range of temperature
from 25 to 80.degree. C. and appeared to have optimal activity at a
temperature of about 60.degree. C.
Temperature stability. A 250 .mu.l sample of each cellobiohydrolase
(EXP02989 or EXP03004) (diluted with 50 mM sodium acetate pH 5
buffer) was incubated at 50, 60 and 70.degree. C. A 20 .mu.l sample
of each cellobiohydrolase was removed and placed on ice at time
points of 0, 10, 30, 60, and 110 minutes. Then 150 .mu.l of 2 g of
PASC per liter of 50 mM sodium acetate pH 5.0 were added to each
sampling, and incubated at 50.degree. C. for 60 minutes. At the end
of the incubation, 50 .mu.l of 2% NaOH were added to stop the
reactions. The reaction mixtures were centrifuged at 1,000.times.g.
The released sugars were measured as described above.
The results shown in FIGS. 10A and 10B indicated that the
cellobiohydrolases were stable at 50.degree. C., less stable at
60.degree. C., and unstable at 70.degree. C. The residual
activities of the EXP02989 cellobiohydrolase were 100% at
50.degree. C., 53% at 60.degree. C., and 40% at 70.degree. C. and
the EXP03004 cellobiohydrolase 100% at 50.degree. C., 57% at
60.degree. C., and 51% at 70.degree. C. after 60 minutes of
incubation.
Deposit of Biological Materials
The following biological materials have been deposited under the
terms of the Budapest Treaty with the Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSM), Mascheroder Weg 1 B,
D-38124 Braunschweig, Germany, and given the following accession
number:
TABLE-US-00004 Strain Accession Number Date E. coli T-CBHII45097
DSM 22598 May 20, 2009 E. coli T-CBHII45178 DSM 22599 May 20,
2009
The strains have been deposited under conditions that assure that
access to the culture will be available during the pendency of this
patent application to one determined by foreign patent laws to be
entitled thereto. The deposits represent substantially pure
cultures of the deposited strains. The deposits are available as
required by foreign patent laws in countries wherein counterparts
of the subject application, or its progeny are filed. However, it
should be understood that the availability of a deposit does not
constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
The invention described and claimed herein is not to be limited in
scope by the specific aspects herein disclosed, since these aspects
are intended as illustrations of several aspects of the invention.
Any equivalent aspects are intended to be within the scope of this
invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims. In the case of conflict, the present disclosure
including definitions will control.
SEQUENCE LISTINGS
1
1211598DNAThielavia hyrcaniae 1atggccaaga agctccttct caccgccgcc
ctcgcggcta ctgccctggc tgctcccatc 60gtcgaggagc gccagaactg tggctccgtc
tggtaagtga ctgcttgatg atctacggtg 120aaagctgctc gctgacacga
cgacttccag gagtcaatgt ggtggccaag ggtggtcggg 180cgccacctgc
tgcgcgtctg gaagcacctg cgtggcccag aacccctggt actcgcagtg
240cctgcccaac agccaggtga ccaccaccgc gactagcgct cgcgcctcgt
cgtcctcgtc 300ctcgtcctcg tccaccaagg cgagcaccag cagcaccagc
cgcaccaccg tgggcaccac 360ctcggtgggc accaccactc gtgctagctc
cacgaccacc tctcccccgg tcgtcacctc 420ggtcgttccc ggcggtgcca
cggccacggc gagctactcg ggcaacccct tctccggcgt 480gcgcctgtgg
gccaacgact actacgcctc cgaggtgtcg actctcgcca tgccttccct
540gacgggcgcc atggccacca aggcggccgc tgtcgccaag gtgcccagct
tccagtggct 600ggaccgcaac gtcaccatcg acaccctgat ggtcaagact
ctgtcccaga tccgcgccgc 660caaccaggcc ggtgccaacc ccccgtatgc
cggtaagttt gagttcgata cttgttcact 720tccaaaagct cggactaacg
acatgtgcag cccagctggt ggtctacgac ctccccgacc 780gtgactgcgc
tgccgccgcc tccaacggcg agttctcgat tgccaacaac ggcgcggcca
840actacaagtc gtacattgac tcgatccgca agcacctcat cgagtactcg
gacatccgca 900ccattctcgt cattgagccc gactcgatgg ccaacatggt
caccaacatg aacgtcgcca 960agtgcagcaa cgcggccacc acgtaccgcg
agctgaccgt ctacgccctc aagcagctga 1020acctgcccca cgtcgccatg
tacctcgacg ccggccacgc cggctggctc ggctggcccg 1080ccaacatcca
gcccgctgcc accctgttcg ccggcatcta cagcgacgct ggcaagcccg
1140cctcggtccg cggtttggcc accaacgtgg ccaactacaa cgcctggagc
ctgtcgtcgg 1200cgccgtcgta cacgagcccc aacgccaact acgacgagaa
gcactacgtc gaggcctttg 1260ccccgctcct ccaggcggcc ggcttccccg
ccaagttcat caccgacacg ggccgcaacg 1320gcaagcagcc cacgggccag
agcgcgtggg gcgactggtg caacgtcaag ggcaccggct 1380tcggtgtccg
cccgacctcg gagacgggcc acgacctcct cgacgccttc gtctgggtca
1440agcccggtgg cgagtcggac ggcaccagcg acaccagcgc cgcccgctac
gactaccact 1500gcggtctgtc ggatgccctc cagcctgctc ccgaggctgg
cacgtggttc caggcctact 1560ttgagcagct gctcaccaac gccaaccctc ctttttag
15982493PRTThielavia hyrcaniae 2Met Ala Lys Lys Leu Leu Leu Thr Ala
Ala Leu Ala Ala Thr Ala Leu1 5 10 15Ala Ala Pro Ile Val Glu Glu Arg
Gln Asn Cys Gly Ser Val Trp Ser 20 25 30Gln Cys Gly Gly Gln Gly Trp
Ser Gly Ala Thr Cys Cys Ala Ser Gly 35 40 45Ser Thr Cys Val Ala Gln
Asn Pro Trp Tyr Ser Gln Cys Leu Pro Asn 50 55 60Ser Gln Val Thr Thr
Thr Ala Thr Ser Ala Arg Ala Ser Ser Ser Ser65 70 75 80Ser Ser Ser
Ser Ser Thr Lys Ala Ser Thr Ser Ser Thr Ser Arg Thr 85 90 95Thr Val
Gly Thr Thr Ser Val Gly Thr Thr Thr Arg Ala Ser Ser Thr 100 105
110Thr Thr Ser Pro Pro Val Val Thr Ser Val Val Pro Gly Gly Ala Thr
115 120 125Ala Thr Ala Ser Tyr Ser Gly Asn Pro Phe Ser Gly Val Arg
Leu Trp 130 135 140Ala Asn Asp Tyr Tyr Ala Ser Glu Val Ser Thr Leu
Ala Met Pro Ser145 150 155 160Leu Thr Gly Ala Met Ala Thr Lys Ala
Ala Ala Val Ala Lys Val Pro 165 170 175Ser Phe Gln Trp Leu Asp Arg
Asn Val Thr Ile Asp Thr Leu Met Val 180 185 190Lys Thr Leu Ser Gln
Ile Arg Ala Ala Asn Gln Ala Gly Ala Asn Pro 195 200 205Pro Tyr Ala
Ala Gln Leu Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 210 215 220Ala
Ala Ala Ala Ser Asn Gly Glu Phe Ser Ile Ala Asn Asn Gly Ala225 230
235 240Ala Asn Tyr Lys Ser Tyr Ile Asp Ser Ile Arg Lys His Leu Ile
Glu 245 250 255Tyr Ser Asp Ile Arg Thr Ile Leu Val Ile Glu Pro Asp
Ser Met Ala 260 265 270Asn Met Val Thr Asn Met Asn Val Ala Lys Cys
Ser Asn Ala Ala Thr 275 280 285Thr Tyr Arg Glu Leu Thr Val Tyr Ala
Leu Lys Gln Leu Asn Leu Pro 290 295 300His Val Ala Met Tyr Leu Asp
Ala Gly His Ala Gly Trp Leu Gly Trp305 310 315 320Pro Ala Asn Ile
Gln Pro Ala Ala Thr Leu Phe Ala Gly Ile Tyr Ser 325 330 335Asp Ala
Gly Lys Pro Ala Ser Val Arg Gly Leu Ala Thr Asn Val Ala 340 345
350Asn Tyr Asn Ala Trp Ser Leu Ser Ser Ala Pro Ser Tyr Thr Ser Pro
355 360 365Asn Ala Asn Tyr Asp Glu Lys His Tyr Val Glu Ala Phe Ala
Pro Leu 370 375 380Leu Gln Ala Ala Gly Phe Pro Ala Lys Phe Ile Thr
Asp Thr Gly Arg385 390 395 400Asn Gly Lys Gln Pro Thr Gly Gln Ser
Ala Trp Gly Asp Trp Cys Asn 405 410 415Val Lys Gly Thr Gly Phe Gly
Val Arg Pro Thr Ser Glu Thr Gly His 420 425 430Asp Leu Leu Asp Ala
Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp 435 440 445Gly Thr Ser
Asp Thr Ser Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu 450 455 460Ser
Asp Ala Leu Gln Pro Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala465 470
475 480Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro Pro Phe 485
49031584DNAThielavia hyrcaniae 3atggccaaga agctccttct caccgccgcc
ctcgcggcct ctgccctggc tgctcccatt 60gtcgaggagc gccagaactg cggctccgtc
tggtaagtga tcgttcgagg attccatggt 120gaaagctgct cgctcacacg
atgctctcca ggagtcaatg tggtggcaac gggtggtcgg 180gcgccacctg
ctgcgcgtct ggaagcacct gcgtagccca gaacccctgg tactcgcagt
240gcctgcccag cagccaggtg accaccacca cgactagcgc tcgcgcctcg
tcgtcgtctt 300cgtcgtccac ccgggcgagc accagcagca ccagccgcac
cacctcggtg ggcaccacca 360cccgtgctag ctccacgacc acctcggccc
ctggcgtcac gtccagcgtt cctggcggtg 420ctacggccac ggccagctac
tcgggcaacc ccttctccgg ggtgcgcctg tgggccaacg 480actactacgc
ctccgaggtg tcgaccctcg ccatgccttc cctgacgggc gccatggcca
540ccaaggcggc cgccgtcgcc aaggtcccca gcttccagtg gctggaccgc
aacgtcacca 600tcgacaccct gatggtcaag actctgtccc agatccgggc
cgccaaccag gccggtgcca 660accccccgta tgccggtaag cttcgactcg
atcttgtctg cctgctctca agtgctcgaa 720ctaagacgat gcgcagccca
gctggtggtc tacgacctcc ccgaccgtga ctgcgctgcc 780gccgcctcca
acggcgagtt ctcgattgcc aacaacggcg cggccaacta caagtcgtac
840atcgactcga tccgcaagca cctcatcgag tactcggaca tccgcaccat
cctggttatt 900gagcccgatt cgatggccaa catggtcacc aacatgaacg
tcgccaagtg cagcaacgcc 960gccacgacct accgcgagct gaccatctac
gctctcaagc agctgaacct gccccacgtc 1020gccatgtacc tcgacgccgg
ccacgccggc tggctcggct ggcccgccaa catccagccc 1080gctgctaccc
tgttcgccgg catctacaac gacgctggca agcccgcctc ggtccgtggt
1140ctggccacca acgtcgccaa ctacaacgcc tggagcctgt cctcggcccc
gtcgtacacg 1200acccccaacg ccaactacga cgagaagcac tacgtcgagg
cctttgcccc gcttctctcg 1260gccgctggct tccccgccaa gttcatcacc
gacactggcc gcaacggcaa gcagcccacc 1320ggccagagcc agtggggcga
ttggtgcaac gtcaagggca ccggcttcgg tgtccgcccg 1380acctccgaga
cgggccacga gctcctggat gcctttgtct gggtcaagcc cggtggcgag
1440tccgacggta ccagcgacac cagcgctgcc cgctacgact accactgcgg
tctgtcggat 1500gccctccagc ccgctcccga ggccggcacg tggttccagg
cctactttga acagctcctc 1560accaacgcca accctccttt ttag
15844487PRTThielavia hyrcaniae 4Met Ala Lys Lys Leu Leu Leu Thr Ala
Ala Leu Ala Ala Ser Ala Leu1 5 10 15Ala Ala Pro Ile Val Glu Glu Arg
Gln Asn Cys Gly Ser Val Trp Ser 20 25 30Gln Cys Gly Gly Asn Gly Trp
Ser Gly Ala Thr Cys Cys Ala Ser Gly 35 40 45Ser Thr Cys Val Ala Gln
Asn Pro Trp Tyr Ser Gln Cys Leu Pro Ser 50 55 60Ser Gln Val Thr Thr
Thr Thr Thr Ser Ala Arg Ala Ser Ser Ser Ser65 70 75 80Ser Ser Ser
Thr Arg Ala Ser Thr Ser Ser Thr Ser Arg Thr Thr Ser 85 90 95Val Gly
Thr Thr Thr Arg Ala Ser Ser Thr Thr Thr Ser Ala Pro Gly 100 105
110Val Thr Ser Ser Val Pro Gly Gly Ala Thr Ala Thr Ala Ser Tyr Ser
115 120 125Gly Asn Pro Phe Ser Gly Val Arg Leu Trp Ala Asn Asp Tyr
Tyr Ala 130 135 140Ser Glu Val Ser Thr Leu Ala Met Pro Ser Leu Thr
Gly Ala Met Ala145 150 155 160Thr Lys Ala Ala Ala Val Ala Lys Val
Pro Ser Phe Gln Trp Leu Asp 165 170 175Arg Asn Val Thr Ile Asp Thr
Leu Met Val Lys Thr Leu Ser Gln Ile 180 185 190Arg Ala Ala Asn Gln
Ala Gly Ala Asn Pro Pro Tyr Ala Ala Gln Leu 195 200 205Val Val Tyr
Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 210 215 220Gly
Glu Phe Ser Ile Ala Asn Asn Gly Ala Ala Asn Tyr Lys Ser Tyr225 230
235 240Ile Asp Ser Ile Arg Lys His Leu Ile Glu Tyr Ser Asp Ile Arg
Thr 245 250 255Ile Leu Val Ile Glu Pro Asp Ser Met Ala Asn Met Val
Thr Asn Met 260 265 270Asn Val Ala Lys Cys Ser Asn Ala Ala Thr Thr
Tyr Arg Glu Leu Thr 275 280 285Ile Tyr Ala Leu Lys Gln Leu Asn Leu
Pro His Val Ala Met Tyr Leu 290 295 300Asp Ala Gly His Ala Gly Trp
Leu Gly Trp Pro Ala Asn Ile Gln Pro305 310 315 320Ala Ala Thr Leu
Phe Ala Gly Ile Tyr Asn Asp Ala Gly Lys Pro Ala 325 330 335Ser Val
Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 340 345
350Leu Ser Ser Ala Pro Ser Tyr Thr Thr Pro Asn Ala Asn Tyr Asp Glu
355 360 365Lys His Tyr Val Glu Ala Phe Ala Pro Leu Leu Ser Ala Ala
Gly Phe 370 375 380Pro Ala Lys Phe Ile Thr Asp Thr Gly Arg Asn Gly
Lys Gln Pro Thr385 390 395 400Gly Gln Ser Gln Trp Gly Asp Trp Cys
Asn Val Lys Gly Thr Gly Phe 405 410 415Gly Val Arg Pro Thr Ser Glu
Thr Gly His Glu Leu Leu Asp Ala Phe 420 425 430Val Trp Val Lys Pro
Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser 435 440 445Ala Ala Arg
Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Gln Pro 450 455 460Ala
Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu465 470
475 480Thr Asn Ala Asn Pro Pro Phe 485525DNAThielavia hyrcaniae
5tgtcctcgat ccgtcgatct gtcgc 25624DNAThielavia hyrcaniae
6tcagccaccg tggcttgtat ccca 24727DNAThielavia hyrcaniae 7aactacgacg
agaagcacta cgtcgag 27824DNAThielavia hyrcaniae 8agctcctgga
tgcctttgtc tgga 24923DNAThielavia hyrcaniae 9aggcgttgta gttggcgacg
ttg 231022DNAThielavia hyrcaniae 10caatcgagaa ctcgccgttg ga
221143DNAThielavia hyrcaniae 11acacaactgg ggatccacca tggccaagaa
gctccttctc acc 431239DNAThielavia hyrcaniae 12gtcaccctct agatctctaa
aaaggagggt tggcgttgg 39
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